IL-I7 homologous polypeptides and therapeutic uses thereof

ABSTRACT

The present invention is directed to novel polypeptides having sequence identity with IL-17 and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention. Further provided herein are methods for treating degenerative cartilaginous disorders.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of novel DNA, therapeutic uses and the recombinant productionof novel polypeptides having sequence identity with the cytokine IL-17,and cytotoxic T-lymphocyte-associated antigen 8 (CTLA-8) designatedherein as PRO1031 and PRO1122 polypeptides.

BACKGROUND OF THE INVENTION

It has been reported that the cytokine interleukin 17 (IL-17) stimulatesepithelial, endothelial, and fibroblastic cells to secrete cytokinessuch as IL-6, IL-8, and granulocyte-colony-stimulating factor, as wellas prostaglandin E2. While expression of IL-17 is restricted toactivated T cells, the IL-17 receptor is widely expressed, a propertyconsistent with the pleiotropic activities of IL-17. Moreover, it hasbeen shown that when cultured in the presence of IL-17, fibroblastscould sustain proliferation of CD34+ preferential maturation intoneutrophils. As a result, IL-17 could be an early potentiator or evenmaintainer of T cell-dependent inflammatory reaction and/or an elementof the cytokine network that bridges the immune system to hematopoiesis.See, Yao, et al., J. Immunol., 155(12):5483-5486 (1995); Fossiez, etal., J. Exp. Med., 183(6):2593-2603 (1996); Kennedy, et al., J.Interferon Cytokine Res., 16(8):611-617(1996).

More generally, all novel proteins are of interest. Extracellularproteins play an important role in the formation, differentiation andmaintenance of multicellular organisms. The fate of many individualcells, e.g., proliferation, migration, differentiation, or interactionwith other cells, is typically governed by information received fromother cells and/or the immediate environment. This information is oftentransmitted by secreted polypeptides (for instance, mitogenic factors,survival factors, cytotoxic factors, differentiation factors,neuropeptides, and hormones) which are, in turn received and interpretedby diverse cell receptors or membrane-bound proteins. These secretedpolypeptides or signaling molecules normally pass through the cellularsecretory pathway to reach their site of action in the extracellularenvironment.

Secreted proteins have various industrial applications, includingpharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents.

Efforts are being undertaken by both industry and academia to identifynew, native secreted proteins. Many efforts are focused on the screeningof mammalian recombinant DNA libraries to identify the coding sequencesfor novel secreted proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci, 93:7108-7113 (1996); U.S. Pat. No.5,536,637)]. The results of such efforts are presented herein.

Interleukin-17 is a recently described, T cell-derived cytokine, thebiological functions of which are only beginning to be understood.Spriggs et al., J. Clin. Immunol. 17: 366 (1997); Broxmeyer, H. E., J.Exp. Med. 183: 2411 (1996). When IL-17 was initially identified as acDNA clone from a rodent T-cell lymphoma, it was recognized as having asequence similar to an open reading frame from a primate herpesvirus,Herpervirus saimiri Rouvier et al., J. Immunol. 150: 5445 (1993), Yao etal., Immunity 3: 811 (1995) [Yao-1], Fossiez et al., J. Exp. Med. 183:2593 (1996). Subsequently, it has been confirmed that this viral proteinhas many if not all of the immunostimulatory activities found for thehost IL-17. Fleckenstein and Desrosiers, “Herpesvirus saimiri andherpesvirus ateles,” In The Herpesviruses, I. B. Roizman, ed, PlenumPublishing Press, New York, p. 253 (1982), Biesinger, B. I. et al.,Procl Natl Acad. Sci. USA 89: 3116 (1992).

Human IL-17 is a 20-30 kDa, disulfide linked, homodimeric protein withvariable glycosylation. Yao-1, supra; Fossier et al, supra. It isencoded by a 155 amino acid open reading frame that includes anN-terminal secretion signal sequence of 19-23 amino acids. The aminoacid sequence of IL-17 is only similar to the Herpesvirus proteindescribed above and does not show significant identity with thesequences of other cytokines or other known proteins. Additionally, theIL-17 encoding mRNA has been detected has only been detected inactivated CD4⁺ memory T cells and PMA/ionomycin stimulated PBMC cells.

Despite its restricted tissue distribution, IL-17 exhibits pleiotropicbiological activities on various types of cells, such as fibroblasts,endothelial cells and epithelial cells. Spriggs, M. K., supra.;Broxmeyer, H. E., supra. IL-17 has been found to stimulate theproduction of many cytokines: TNF-α and IL-1β from macrophages[Jovanovic et al., J. Immunol 160: 3513 (1998)]; IL-6, IL-8 and theintracellular adhesion molecule (ICAM-1) from human fibroblasts. Fossiezet al., supra, Yao et al., J. Immunol. 155: 5483 (1995) [Yao-2];granulocyte-colony-stimulating factor (G-CSM) and prostaglandin (PGE-2)form synoviocytes, Fossiez et al., supra. Through the induction of anumber of cytokines, IL-17 is able to mediate a wide-range of response,mostly proinflammatory and hematopoietic. This has led to the suggestionthat IL-17 may play a pivotal role in initiating or sustaining aninflammatory response. Jovanovic et al., supra.

Consistent with IL-17's wide-range of effects, the cell surface receptorfor IL-17 has been found to be widely expressed in many tissues and celltypes Yao et al., Cytokine 9: 794 (1997) [Yao-3]. While the amino acidsequence of the hIL-17 receptor (866 a.a.) predicts a protein with asingle transmembrane domain and a long, 525 amino acid intracellulardomain, the receptor sequence is unique and is not similar to that ofany of the receptor from the cytokine/growth factor receptor family.This coupled with the lack of similarity of IL-17 itself to other knownproteins indicates that IL-17 and its receptor may be part of a novelfamily of signaling proteins and receptors.

IL-17 has further been shown, by intracellular signaling, to stimulatetransient Ca²⁺ influx and a reduction in [cAMP]_(i) in humanmacrophages. Jovanovic et al., supra. Fibroblasts and macrophagestreated with IL-17 induce the activation of NF-κB, Yao-1, supra,Jovanovic et al, supra., while macrophages treated with it activateNF-κB and mitogen-activated protein kinases. Shalom-Barek et al., J.Biol. Chem. 273: 27467 (1998).

The present invention describes the cloning and characterization of twonovel proteins, termed PRO1031 (IL-17B) and PRO1122 (IL-17C), and activevariants thereof, that are similar in amino acid sequence to IL-17.

SUMMARY OF THE INVENTION

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving sequence identity with IL-17, wherein the polypeptide isdesignated in the present application as “PRO1031” or “PRO1122”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1031 or PRO1122 polypeptide.

In one aspect, the isolated nucleic acid comprises DNA encoding thePRO1031 or PRO1122 polypeptide having amino acid residues: from about 21through 180 of FIG. 1 (SEQ ID NO:1), or from about 19 through 197 ofFIG. 3 (SEQ ID NO:3), respectively, or is complementary to such encodingnucleic acid sequence, and remains stably bound to it under at leastmoderate, and optionally, under high stringency conditions.

In another embodiment, the isolated nucleic acid comprises DNA having atleast about 80% sequence identity, preferably at least about 81%sequence identity, more preferably at least about 82% sequence identity,yet more preferably at least about 83% sequence identity, yet morepreferably at least about 84% sequence identity, yet more preferably atleast about 85% sequence identity, yet more preferably at least about86% sequence identity, yet more preferably at least about 87% sequenceidentity, yet more preferably at least about 88% sequence identity, yetmore preferably at least about 89% sequence identity, yet morepreferably at least about 90% sequence identity, yet more preferably atleast about 91% sequence identity, yet more preferably at least about92% sequence identity, yet more preferably at least about 93% sequenceidentity, yet more preferably at least about 94% sequence identity, yetmore preferably at least about 95% sequence identity, yet morepreferably at least about 96% sequence identity, yet more preferably atleast about 97% sequence identity, yet more preferably at least about98% sequence identity, yet more preferably at least about 99% sequenceidentity to (a) a DNA molecule encoding a PRO1031 or PRO1122 polypeptidecomprising the sequence of amino acid residues from 1 or about 21 to180, inclusive, of FIG. 1 (SEQ ID NO:1) or from 1 or about 19 to 197,inclusive, of FIG. 3 (SEQ ID NO:3), or the (b) the complement of the DNAmolecule of (a). Alternatively, the isolated nucleic acid comprises DNAencoding the PRO1031 polypeptide having amino acid residues 1 through180, inclusive, of FIG. 3 (SEQ ID NO:3). Alternatively, the isolatednucleic acid comprises DNA encoding a 1122 polypeptide having thesequence of amino acid residues from about 1 to about 197, inclusive ofFIG. 1 (SEQ ID NO:1).

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding a PRO1031 or PRO1122 polypeptide comprising DNAhybridizing to the complement of the nucleic acid between aboutresidues: (a) 42 to about 581, inclusive, of FIG. 2 (SEQ ID NO:2), or(b) 49 to about 640, inclusive, or FIG. 4 (SEQ ID NO:4), respectively.Preferably, the hybridization range extends from about nucleic acidresidue (a) about 102 to about 581, inclusive, of FIG. 2 (SEQ ID NO:2),or (b) about 104 to about 640, inclusive, of FIG. 4 (SEQ ID NO:4),respectively. Preferably, hybridization occurs under stringenthybridization and wash conditions.

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding an active PRO1031 or PRO1122 polypeptide comprising anucleotide sequence that hybridizes to the complement of a nucleic acidsequence that encodes amino acids (a) 1 or about 21 to about 180,inclusive, of FIG. 1 (SEQ ID NO:1), or (b) 1 or about 19 to about 197,inclusive, of FIG. 3 (SEQ ID NO:3). Preferably, hybridization occursunder stringent hybridization and wash conditions.

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA having at least about 80% sequence identity,preferably at least about 81% sequence identity, more preferably atleast about 82% sequence identity, yet more preferably at least about83% sequence identity, yet more preferably at least about 84% sequenceidentity, yet more preferably at least about 85% sequence identity, yetmore preferably at least about 86% sequence identity, yet morepreferably at least about 87% sequence identity, yet more preferably atleast about 88% sequence identity, yet more preferably at least about89% sequence identity, yet more preferably at least about 90% sequenceidentity, yet more preferably at least about 91% sequence identity, yetmore preferably at least about 92% sequence identity, yet morepreferably at least about 93% sequence identity, yet more preferably atleast about 94% sequence identity, yet more preferably at least about95% sequence identity, yet more preferably at least about 96% sequenceidentity, yet more preferably at least about 97% sequence identity, yetmore preferably at least about 98% sequence identity, yet morepreferably at least about 99% sequence identity to (a) a DNA moleculeencoding the same mature polypeptide encoded by the human protein cDNAin ATCC deposit No. 209866 (DNA59294-1381) or 203552 (DNA62377-1381-1).In a preferred embodiment, the nucleic acid comprises DNA encoding thesame mature polypeptide encoded by the human protein cDNA in ATCCdeposit number 209866 (DNA59294-1381) or 203553 (DNA62377-1381-1),deposited on 14 May 1998 and 23 Dec. 1998, respectively. In a morepreferred embodiment, the nucleic acid comprises the cDNA insert of ATCCdeposit DNA59294-1381 (ATCC 209866) deposited on 14 May 1998 orDNA62377-1381-1 (ATCC 203552), deposited on 22 Dec. 1998, respectively.

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a protein having atleast about 80% sequence identity, preferably at least about 81%sequence identity, more preferably at least about 82% sequence identity,yet more preferably at least about 83% sequence identity, yet morepreferably at least about 84% sequence identity, yet more preferably atleast about 85% sequence identity, yet more preferably at least about86% sequence identity, yet more preferably at least about 87% sequenceidentity, yet more preferably at least about 88% sequence identity, yetmore preferably at least about 89% sequence identity, yet morepreferably at least about 90% sequence identity, yet more preferably atleast about 91% sequence identity, yet more preferably at least about92% sequence identity, yet more preferably at least about 93% sequenceidentity, yet more preferably at least about 94% sequence identity, yetmore preferably at least about 95% sequence identity, yet morepreferably at least about 96% sequence identity, yet more preferably atleast about 97% sequence identity, yet more preferably at least about98% sequence identity, yet more preferably at least about 99% sequenceidentity to (a) the full-length polypeptide encoded by the cDNAdeposited with the ATCC on (1) 14 May 1998 under ATCC Deposit No.:209866 (DNA59294-1381) or (2) 23 Dec. 1998 under ATCC Deposit No.:203553 (DNA62377-1381-1), or (b) the complement of the nucleotidesequence of (a). In a preferred embodiment, the isolated nucleic acidmolecule encodes the same full length polypeptide as the cDNA deposit ofATCC Deposit No.: 209866 or 203553, respectively.

In a further aspect, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% sequence identity,preferably at least about 81% sequence identity, more preferably atleast about 82% sequence identity, yet more preferably at least about83% sequence identity, yet more preferably at least about 84% sequenceidentity, yet more preferably at least about 85% sequence identity, yetmore preferably at least about 86% sequence identity, yet morepreferably at least about 87% sequence identity, yet more preferably atleast about 88% sequence identity, yet more preferably at least about89% sequence identity, yet more preferably at least about 90% sequenceidentity, yet more preferably at least about 91% sequence identity, yetmore preferably at least about 92% sequence identity, yet morepreferably at least about 92% sequence identity, yet more preferably atleast about 93% sequence identity, yet more preferably at least about94% sequence identity, yet more preferably at least about 95% sequenceidentity, yet more preferably at least about 96% sequence identity, yetmore preferably at least about 97% sequence identity, yet morepreferably at least about 98% sequence identity, yet more preferably atleast about 99% sequence identity, to: (a) DNA molecule comprising thesequence of nucleotides from about 42 or about 102 to about 581,inclusive, of FIG. 2 (SEQ ID NO:2) or from about 49 or about 104 toabout 640, inclusive, of FIG. 4 (SEQ ID NO:4); or (b) the complement ofthe DNA molecule of (a).

In another aspect, the isolated nucleic acid molecule comprises: (a) thenucleotide sequence from about 42 or about 102 to about 581, inclusive,of FIG. 2 (SEQ ID NO:2) or from about 49 or about 104 to about 640,inclusive, of FIG. 4 (SEQ ID NO:4); or (b) the complement of the DNAmolecule of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule produced by hybridizing a test DNA molecule under stringentconditions with: (a) a DNA molecule encoding (i) a PRO1031 polypeptidehaving the sequence of amino acid residues from about 1 or about 21 toabout 180, inclusive, of FIG. 1 (SEQ ID NO:1), or (ii) a PRO1122polypeptide having the sequence of amino acid residues from about 1 orabout 19 to about 197, inclusive, of FIG. 3 (SEQ ID NO:3); or (b) thecomplement of the DNA molecule of (a), and if the DNA molecule has atleast about an 80% sequence identity, preferably at least about an 81%sequence identity, more preferably at least about a 82% sequenceidentity, yet more preferably at least about a 83% sequence identity,yet more preferably at least about 84% sequence identity, yet morepreferably at least about 85% sequence identity, yet more preferably atleast about 86% sequence identity, yet more preferably at least about87% sequence identity, yet more preferably at least about 88% sequenceidentity, yet more preferably at least about 89% sequence identity, yetmore preferably at least about 90% sequence identity, yet morepreferably at least about 91% sequence identity, yet more preferably atleast about 92% sequence identity, yet more preferably at least about93% sequence identity, yet more preferably at least about 94% sequenceidentity, yet more preferably at least about 95% sequence identity, yetmore preferably at least about 96% sequence identity, yet morepreferably at least about 97% sequence identity, yet more preferably atleast about 98% sequence identity, yet more preferably at least about99% sequence identity to (a) or (b), isolating the test DNA molecule.

In yet a further aspect, the invention concerns an isolated nucleic acidmolecule comprising: (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 81% positives, morepreferably at least about 82% positives, yet more preferably at leastabout 83% positives, yet more preferably at least about 84% positives,yet more preferably at least about 85% positives, yet more preferably atleast about 86% positives, yet more preferably at least about 87%positives, yet more preferably at least about 88% positives, yet morepreferably at least about 89% positives, yet more preferably at leastabout 90% positives, yet more preferably at least about 91% positives,yet more preferably at least about 92% positives, yet more preferably atleast about 93% positives, yet more preferably at least about 94%positives, yet more preferably at least about 95% positives, yet morepreferably at least about 96% positives, yet more preferably at leastabout 97% positives, yet more preferably at least about 98% positives,yet more preferably at least about 99% positives, when compared with theamino acid sequence of residues about (i) 21 to about 180, inclusive, ofFIG. 1 (SEQ ID NO:1), or (ii) 19 to about 197, inclusive, of FIG. 3 (SEQID NO:3), or (b) the complement of the DNA of (a).

In a specific aspect, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO1032 or PRO1122 polypeptidewithout the N-terminal signal sequence and/or initiating methionine, oris complementary to such encoding nucleic acid molecule. The signalpeptide has been tentatively identified as extending from about aminoacid residue (a) 1 to about amino acid residue 20, inclusive, in thesequence of FIG. 1 (SEQ ID NO:1), or (b) 1 to about amino acid residue18, inclusive, in the sequence of FIG. 3 (SEQ ID NO:3). It is noted,however, that the C-terminal boundary of the signal peptide may vary,but most likely by no more than about 5 amino acids on either side ofthe signal peptide C-terminal boundary as initially identified herein,wherein the C-terminal boundary of the signal peptide may be identifiedpursuant to criteria routinely employed in the art. Nielsen et al.,Prot. Engin. 10: 1-6 (1997), von Heinje et al., Nucl. Acids Res. 14:4683-4690 (1986). Moreover, it is also recognized that, in some cases,cleavage of the signal sequence form a secreted polypeptide is notentirely uniform, resulting in more than one secreted species. Thesepolypeptides, and the polynucleotides encoding them, are contemplated bythe present invention. A such, for purposed of the present application,the signal peptide of the PRO1032 or PRO1122 polypeptide shown in FIG. 1(SEQ ID NO:1) or FIG. 3 (SEQ ID NO:3), respectively, extends from aminoacids 1 to X, wherein X is any amino acid from (a) 15 to 25 of FIG. 1(SEQ ID NO:1), or (b) 13 to 23 of FIG. 3 (SEQ ID NO:3), respectively ofFIG. 3.

Another embodiment is directed to fragments of a PRO1031- orPRO1122-encoding sequence that may find use as, for example,hybridization probes or for encoding fragments of a PRO1031 or PRO1122polypeptide that may optionally encode a polypeptide comprising abinding site for an anti-PRO1031 or anti-PRO1122 antibody. Such nucleicacids fragments are usually at least about 20 nucleotides in length,preferably at least about 30 nucleotides in length, more preferable atleast about 40 nucleotides in length, yet more preferably at least about50 nucleotides in length, yet more preferably at least about 60nucleotides in length, yet more preferably at least about 70 nucleotidesin length, yet more preferably at least about 80 nucleotides in length,yet more preferably at least about 90 nucleotides in length, yet morepreferably at least about 100 nucleotides in length, yet more preferablyat least about 110 nucleotides in length, yet more preferably at leastabout 120 nucleotides in length, yet more preferably at least about 130nucleotides in length, yet more preferably at least about 140nucleotides in length, yet more preferably at least about 150nucleotides in length, yet more preferably at least about 160nucleotides in length, yet more preferably at least about 170nucleotides in length, yet more preferably at least about 180nucleotides in length, yet more preferably at least about 190nucleotides in length, yet more preferably at least about 200nucleotides in length, yet more preferably at least about 250nucleotides in length, yet more preferably at least about 300nucleotides in length, yet more preferably at least about 350nucleotides in length, yet more preferably at least about 400nucleotides in length, yet more preferably at least about 450nucleotides in length, yet more preferably at least about 500nucleotides in length, yet more preferably at least about 600nucleotides in length, yet more preferably at least about 700nucleotides in length, yet more preferably at least about 800nucleotides in length, yet more preferably at least about 900nucleotides in length, yet more preferably at least about 100nucleotides in length, wherein in this context “about” means thereferenced nucleotide sequence length plus or minus 10% of thatreferenced length. In a preferred embodiment, the nucleotide sequencefragment is derived from any coding region of the nucleotide sequenceshown in FIG. 2 (SEQ ID NO:2) or FIG. 4 (SEQ ID NO:4). In a morepreferred embodiment, the nucleotide sequence fragment is derived fromnucleotides about 50 to about 390 and about 621 through about 640,inclusive, of FIG. 4 (SEQ ID NO:4). Alternatively, the nucleotidesequence fragment can be derived from a fragment within the regionbetween 391 and 620, inclusive, provided at least one nucleotide isincluded outside of the region (i.e., 50-390, 621-640).

In another embodiment, the invention provides a vector comprising DNAencoding a PRO1031 or PRO1122 or its variants. The vector may compriseany of the isolated nucleic acid molecules hereinabove defined.

In another embodiment, the invention provides a host cell comprising theabove vector. By way of example, the host cells may be CHO cells, E.coli, or yeast. A process for producing PRO1031 or PRO1122 polypeptidesis further provided and comprises culturing host cells under conditionssuitable for expression of PRO1031 or PRO1122, respectively, andrecovering PRO1031 or PRO1122, respectively, from the cell culture.

In another embodiment, the invention provides isolated PRO1031 orPRO1122 polypeptides encoded by any of the isolated nucleic acidsequences hereinabove defined.

In another aspect, the invention concerns an isolated PRO1031 or PRO1122polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 81% sequence identity, morepreferably about 82% sequence identity, yet more preferably at leastabout 83% sequence identity, yet more preferably at least about 84%sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity, yetmore preferably at least about 99% sequence identity to the sequence ofamino acid residues about (a) 1 or about 21 to about 180, inclusive, ofFIG. 1 (SEQ ID NO:1), or (b) 1 or about 19 to about 197, inclusive, ofFIG. 3 (SEQ ID NO:3), respectively. In a preferred aspect, thepolypeptide comprises amino acid residues about (a) 1 or about 21 toabout 180, inclusive, of FIG. 1 (SEQ ID NO:1) or (b) 1 or about 19 toabout 197, inclusive, of FIG. 3 (SEQ ID NO:3), respectively.

In a further aspect, the invention concerns an isolated PRO1031 orPRO1122 polypeptide comprising an amino acid sequence having at leastabout 80% sequence identity, preferably at least about 81% sequenceidentity, more preferably at least about 82% sequence identity, yet morepreferably at least about 83% sequence identity, yet more preferably atleast about 84% sequence identity, yet more preferably at least about85% sequence identity, yet more preferably at least about 86% sequenceidentity, yet more preferably at least about 87% sequence identity, yetmore preferably at least about 88% sequence identity, yet morepreferably at least about 89% sequence identity, yet more preferably atleast about 90% sequence identity, yet more preferably at least about91% sequence identity, yet more preferably at least about 92% sequenceidentity, yet more preferably at least about 93% sequence identity, yetmore preferably at least about 94% sequence identity, yet morepreferably at least about 95% sequence identity, yet more preferably atleast about 96% sequence identity, yet more preferably at least about97% sequence identity, yet more preferably at least about 98% sequenceidentity, yet more preferably at least about 99% sequence identity tothe amino acid encoded by the human protein cDNA deposited with the ATCCon (1) 14 May 1999 under ATCC Deposit No. 209866 (DNA59294-1381) or (2)22 Dec. 1998 under ATCC Deposit No. 203552, respectively.

In a preferred embodiment, the PRO1031 or PRO1122 polypeptide isobtained or obtainable by expressing the polypeptide encoded by the cDNAinsert of the vector deposited on (a) 14 May 1998 under ATCC depositnumber 209866 (DNA59294-1381), or (b) 22 Dec. 1998 under ATCC depositnumber 203552 (DNA62377-1381-1).

In a further aspect, the invention concerns an isolated PRO1031 orPRO1122 polypeptide comprising an amino acid sequence scoring at leastabout 80% positives, preferably at least about 81% positives, morepreferably at least about 82% positives, yet more preferably at leastabout 83% positive, yet more preferably at least about 84% positives,yet more preferably at least about 85% positives, yet more preferably atleast about 86% positives, yet more preferably at least about 87%positives, yet more preferably at least about 88% positives, yet morepreferably at least about 89% positives, yet more preferably at leastabout 90% positives, yet more preferably at least about 91% positives,yet more preferably at least about 92% positives, yet more preferably atleast about 93% positives, yet more preferably at least about 94%positives, yet more preferably at least about 95% positives, yet morepreferably at least about 96% positives, yet more preferably at leastabout 97% positives, yet more preferably at least about 98% positives,yet more preferably at least about 99% positives, when compared with theamino acid sequence of residues from about (1) 1 or about 21 to about180, inclusive, of FIG. 1 (SEQ ID NO:1), or (2) 1 or about 19 to about197, inclusive, of FIG. 3 (SEQ ID NO:3).

In a specific aspect, the invention provides an isolated PRO1031 orPRO1122 polypeptide without the N-terminal signal sequence and/orinitiating methionine and is encoded by a nucleotide sequence thatencodes such an amino acid sequence as hereinbefore described. Processesfor producing the same are also herein described, wherein thoseprocesses comprise culturing a host cell comprising a vector whichcomprises the appropriate encoding nucleic acid molecule underconditions suitable for expression of the PRO1031 or PRO1122 polypeptideand recovering the PRO1031 or PRO1122 polypeptide, respectively, fromthe cell culture.

In still a further aspect, the invention provides a polypeptide producedby: (1) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding a (i) PRO1031 polypeptide having thesequence of amino acid residues from about 21 to about 180, inclusive,of FIG. 1 (SEQ ID NO:1), or (ii) PRO1122 polypeptide having the sequenceof amino acid residues from about 19 to about 197, inclusive, of FIG. 3(SEQ ID NO:3), or (b) the complement of the DNA molecule of (a); and ifthe test DNA molecule has at least about an 80% sequence identity,preferably at least about an 81% sequence identity, more preferably atleast about an 82% sequence identity, yet more preferably at least aboutan 83% sequence identity, yet more preferably at least about an 84%sequence identity, yet more preferably at least about an 85% sequenceidentity, yet more preferably at least about an 86% sequence identity,yet more preferably at least about an 87% sequence identity, yet morepreferably at least about an 88% sequence identity, yet more preferablyat least about an 89% sequence identity, yet more preferably at leastabout a 90% sequence identity, yet more preferably at least about a 91%sequence identity, yet more preferably at least about a 92% sequenceidentity, yet more preferably at least about a 93% sequence identity,yet more preferably at least about a 94% sequence identity, yet morepreferably at least about a 95% sequence identity, yet more preferablyat least about a 96% sequence identity, yet more preferably at leastabout a 97% sequence identity, yet more preferably at least about 98%sequence identity, yet more preferably at least about a 99% sequenceidentity to (a) or (b); (2) culturing a host cell comprising the testDNA molecule under conditions suitable for expression of thepolypeptide, and (3) recovering the polypeptide from the cell culture.

In yet another aspect, the invention concerns an isolated PRO1031 orPRO1122 polypeptide comprising the sequence of amino acid residues fromabout (1) 1 or about 21 to about 180, inclusive, of FIG. 1 (SEQ IDNO:1), or (2) 1 or about 19 to about 197, inclusive, of FIG. 3 (SEQ IDNO:3), respectively, or a fragment thereof which is biologically activeor sufficient to provide a binding site for an anti-PRO1031 oranti-PRO1122 antibody, respectively, wherein the identification ofPRO1031 or PRO1122 polypeptide fragments, respectively, that possessbiological activity or provide a binding site for an anti-PRO1031 oranti-PRO1122 antibody, respectively, may be accomplished in a routinemanner using techniques which are well known in the art.

In another embodiment, the invention provides chimeric moleculescomprising a PRO1031 or PRO1122 polypeptide fused to a heterologouspolypeptide or amino acid sequence. An example of such a chimericmolecule comprises a PRO1031 or PRO1122 polypeptide, respectively, fusedto an epitope tag sequence or a Fc region of an immunoglobulin.

In another embodiment, the invention provides an antibody whichspecifically binds to a PRO1031 or PRO1122 polypeptide. Optionally, theantibody is a monoclonal antibody, an antibody fragment or a singlechain antibody.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO1031 or PRO1122 polypeptide. In a particularaspect, the agonist or antagonist is an anti-PRO1031 or anti-PRO1122antibody, or a small molecule.

In yet another embodiment, the invention concerns a method ofidentifying agonists or antagonists of a native PRO1031 or nativePRO1122 polypeptide, by contacting the native PRO1031 or PRO1122polypeptide with a candidate molecule and monitoring a biologicalactivity mediated by said polypeptide.

In still a further embodiment, the invention concerns a compositioncomprising a PRO1031 or PRO1122 polypeptide, or an agonist or antagonistas hereinabove defined, in combination with a carrier. Preferably, thecarrier is pharmaceutically acceptable.

In still a further embodiment, the invention concerns the use of aPRO1031 or PRO1122 polypeptide, or an agonist or antagonist thereof ashereinbefore described, or an anti-PRO1031 or anti-PRO1122 antibody, forthe preparation of a medicament useful in the treatment of a conditionwhich is responsive to the PRO1031 or PRO1122 polypeptide or an agonistor antagonist thereof (e.g., anti-PRO1031 or PRO1122). In a particularaspect, the invention concerns the use of a PRO1031 or PRO1122polypeptide, or an agonist or antagonist thereof in a method fortreating a degenerative cartilaginous disorder.

In still a further embodiment, the invention relates to a method oftreating a degenerative cartilaginous disorder by administration of atherapeutically effective amount of a PRO1031 or PRO1122 polypeptide,agonist, or antagonist thereof to a mammal suffering from said disorder.

In still a further embodiment, the invention relates to a method ofdiagnosing a degenerative cartilaginous disorder by (1) culturing testcells or tissues expressing PRO1031 or PRO1122; (2) administering acompound which can inhibit PRO1031 or PRO1122 modulated signaling; and(3) measuring the PRO1031 or PRO1122 mediated phenotypic effects in thetest cells.

In still a further embodiment, the invention relates to PRO1031 orPRO1122 antagonists and/or agonist molecules. In one aspect, theinventions provides a method of screening compounds which mimic PRO1031or PRO1122 (agonists) or diminish the effect of the PRO1031 or PRO1122(antagonists).

In still a further embodiment, the invention relates to a therapeuticcomposition comprising a therapeutically effective amount of PRO1031,PRO1122, antagonist or agonist thereof in combination with apharmaceutically-acceptable carrier.

In still a further embodiment, the invention relates to an article ofmanufacture comprising a container, label and therapeutically effectiveamount of PRO1031, PRO1122, antagonist or agonist thereof in combinationwith a pharmaceutically-acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence (SEQ ID NO:1) derived fromnucleotides 42-581 of SEQ ID NO:2. Also shown in FIG. 1 are the signalpeptide, an N-glycosylation site, a region having sequence identity withIL-17, the molecular weight, and approximate pI.

FIG. 2 shows a nucleotide sequence (SEQ ID NO:2) containing thenucleotide sequence of a native sequence PRO1031 cDNA (nucleotides42-581 of SEQ ID NO:2), wherein the nucleotide sequence (SEQ ID NO:2) isa clone designated herein as “UNQ516” and/or “DNA59294-1381”.

FIG. 3 shows the amino acid sequence (SEQ ID NO:3) derived fromnucleotides 50-640 of SEQ ID NO:4. Also shown are the approximatelocations of the signal peptide, a leucine zipper pattern in a regionhaving sequence identity with IL-17. The approximate weight in daltons,and approximate pI are also shown.

FIG. 4 shows a nucleotide sequence (SEQ ID NO:4) containing thenucleotide sequence of a native sequence PRO1122 cDNA (nucleotides50-640 of SEQ ID NO:1), wherein the nucleotide sequence (SEQ ID NO:4) isa clone designated herein as “UNQ561” and/or “DNA62377-1381-1”. Thecomplementary sequence and deduced amino acid sequences are also shown.

FIG. 5 shows DNA47332 (SEQ ID NO:5), a virtual DNA fragments used in theisolation of DNA59294 (SEQ ID NO:2).

FIG. 6 shows DNA49665 (Incyte EST 1347523) (SEQ ID NO:7), a virtualfragment used in the isolation of DNA62377 (SEQ ID NO:4).

FIG. 7A shows an alignment between the protein sequences encoded byDNA59624 (IL17-B) (SEQ ID NO:1), DNA62377 (IL17-C) (SEQ ID NO:3) andIL-17 (SEQ ID NO:11). The putative signal sequences are underlined,potential N-linked glycosylation sites are double underlined, andconserved tryptophan and cysteine residues are marked with asterisks.IL-17, IL-17B and IL-17C share 26-28% amino acid identity with eachother. FIG. 7B shows an alignment between just the encoded protein fromDNA59624 (SEQ ID NO:1) and DNA62377 (SEQ ID NO:3).

FIG. 8 is an RNA blot analysis of IL-17B (UNQ516) (SEQ ID NO:1). Thenorthern blot depicts mRNA from human tissues (Clontech) hybridized to ahuman IL17B specific radiolabeled probe as described in Example 8. RNAsize markers are shown on the left. A rehybridization of the same blotwith a human β-actin cDNA probe is shown at the bottom.

FIGS. 9A-9B depict bar graphs representing the biological activities ofIL17 (SEQ ID NO:11), IL17B (UNQ516) (SEQ ID NO:1) and IL17C (UNQ561)(SEQ ID NO:3). FIG. 9A shows human foreskin fibroblast (HFF) cellscultured with control Fc fusion protein, IL-17, IL-17B.Fc (SEQ ID NO:12)or IL-17C.Fc (SEQ ID NO:13) at 100 ng/ml for 18 hours and theconditioned media were assayed for IL-6 (SEQ ID NO:14) as described inExample 10. FIG. 9B shows the human leukemic cell line, THP1, which wastreated with the same cytokines (100 ng/ml) as above under the sameconditions wherein the supernatants were assayed for the level of TNF-αrelease. Results are expressed as the mean+/−SE of triplicatedeterminations from one representative experiment.

FIG. 10 is a time course representing the dependence of IL17B and IL17Cactivated TNF-α release from THP1 cells. In FIG. 10A, THP1 cells wereincubated with 100 ng/ml (2.2 nM) of IL17B.Fc (SEQ ID NO:12) or IL17C.Fc(SEQ ID NO:13) for 0.5 to 32 hours, the conditioned media harvested, andthe TNF-α concentration quantitated as described in Example 10. In FIG.10B, THP1 cells were treated with the IL-17B.Fc and IL-17C.Fc at aconcentration range from 0 to 120 nM for 18 hours and the TNF-α releasedetermined.

FIG. 11 is an immunoprecipitation of IL-17R ECD (SEQ ID NO:15) withIL-17 (SEQ ID NO:11), IL17B (SEQ ID NO:1) and IL-17C (SEQ ID NO:3).His-tagged IL-17 receptor ECD was expressed in 293 cells andmetabolically labeled with ³⁵S as described in Example 11. Thesupernatant was recovered and Ni—NTA beads were used to affinityprecipitate the his-tagged IL-17R ECD (SEQ ID NO:15) in the supernatant(lane 1). In FIG. 11A, IL-17 (SEQ ID NO:11), IL-17B.Fc (SEQ ID NO:12)and IL-17C.Fc (SEQ ID NO:13), or control Fc fusion proteins wereincubated with the supernatant and protein-A-agarose beads were added toprecipitate the Fc fusion proteins. For the IL-17 immunoprecipitationreaction, anti-IL-17 antibodies were included. FIG. 11B shows theresults of a competitive binding experiment, wherein immunoprecipitationof IL-17R ECD (SEQ ID NO:22) by IL-17 (SEQ ID NO:11) was performed inthe presence of a five-fold excess of IL-17B.his (SEQ ID NO:23) andcontrol his-tagged proteins. Precipitates in both FIG. 11A and FIG. 11Bwere analyzed by electrophoresis on NuPAGE (4-12% Bis-Tris) gels.Molecular weight markers are indicated on the left of each panel.

FIG. 12 shows FACS analysis of the binding of IL-17B.Fc (SEQ ID NO:12)and IL-17C.Fc (SEQ ID NO:13) to THP-1 cells. THP-1 cells were incubatedwith IL-17B.Fc (A) or IL-17C.Fc (B) or control Fc fusion proteins in PBS(5% horse serum) and followed by addition of FITC conjugated anti-Fcsecondary antibodies.

FIG. 13 shows the effect of IL-17 (SEQ ID NO:11) on articular cartilage.Cartilage explants were cultured with the indicated concentration ofIL-17 alone (solid) or in the presence of IL-1α at the indicatedconcentration (hatched) (SEQ ID NO:25) or IL1ra (IL-1 receptorantagonist, R&D Systems, 1 μg/ml) (SEQ ID NO:26) for 72 hours. Releaseof proteolycans (PG) into the media (top panel) indicates matrixbreakdown. Matrix synthesis was determined by incorporation of³⁵S-sulphate into the tissue (bottom panel).

FIG. 14 shows the effect of IL-17 (SEQ ID NO:11) on the release ofnitric oxide. Explants were treated with IL-17 (10 ng/ml) alone (leftcolumns) or in the presence of IL-1α (10 ng/ml) (SEQ ID NO:25) (rightcolumns). After 48 hours, media was assayed for nitrite concentration.

FIG. 15 shows the effect of NO on IL-17 induced changes in matrixmetabolism. Explants were treated with IL-17 (5 ng/ml) (SEQ ID NO:11)alone (+) or with an irreversible inhibitor of nitric oxide synthase,NOS (L-NIO, Caymen Chemical, 0.5 mM). After 72 hours of treatment, mediawas assayed for (A) nitrite and (B) proteoglycans (PGs). (C)Proteoglycan synthesis was determined by incorporation of ³⁵S-sulphateinto the tissue.

FIG. 16 shows the effect of the inhibition of NO on IL-1α-inducedchanges in proteoglycan (PG) metabolism. Articular cartilage explantswere treated with IL-1α (5 ng/ml) (SEQ ID NO:25) alone (+) or withinhibitors of NOS (L-NIO or L-NIL) (L-NIL, reversible NOS inhibitor,Caymen Chemical) or IL-1ra (IL-1 receptor antagonist, R&D Systems, 1Ξg/ml) (SEQ ID NO:26). After 72 hours or treatment, media as assayed for(A) nitrite concentration and (B) amount of proteoglycans. (C) Matrixsynthesis was determined by incorporation of ³⁵S-sulphate into thetissue.

FIG. 17 shows the effect of UNQ516 (SEQ ID NO:1) on articular cartilage.Explants were treated with UNQ561 at 1% or 0.1% in the absence (leftmost3 columns) or presence (rightmost three columns) of IL-1α (SEQ ID NO:25)at 10 ng/ml, and proteoglycan (PG) synthesis and nitrite production weredetermined as described in Example 16.

FIG. 18 shows the effect of UNQ561 (SEQ ID NO:3) on articular cartilage.Explants were treated with UNQ561 at 1% or 0.1% in the absence (leftmostthree columns) or presence (rightmost three columns) of IL-1α (+) (10ng/ml) (SEQ ID NO:25). Proteoglycan (PG) release and synthesis are shownas amount above control.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

The terms “PRO1031 polypeptide”, or “PRO1122 polypeptide” and “PRO1031”,or “PRO1122” when used herein encompass native sequence PRO1031, nativesequence PRO1122, respectively and polypeptide variants thereof (whichare further defined herein). The PRO1031 or PRO1122 polypeptides may beisolated from a variety of sources, such as from human tissue types orfrom another source, or prepared by recombinant or synthetic methods.

A “native sequence PRO1031 polypeptide” or “native sequence PRO1122polypeptide” comprise a polypeptide having the same amino acid sequenceas a PRO1031 or PRO1122 polypeptide, respectively, derived from nature.Such native sequence PRO1031 or PRO1122 polypeptide can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence PRO1031 polypeptide” or “native sequence PRO1122polypeptide” specifically encompasses naturally-occurring truncated orsecreted forms of a PRO1031 polypeptide or PRO1122 polypeptide,respectively, (e.g., soluble forms containing for instance, anextracellular domain sequence), naturally-occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants ofa PRO1031 or PRO1122 polypeptide, respectively.

In one embodiment of the invention, the native sequence PRO1031polypeptide or PRO1122 polypeptide is a full-length or mature nativesequence (a) PRO1031 polypeptide comprising amino acids 1 or 21 through180 of FIG. 1 (SEQ ID NO:1) or (b) PRO1122 polypeptide comprising aminoacids 1 or 19 through 197 of FIG. 3 (SEQ ID NO:3), respectively. Also,while the PRO1031 or PRO1122 polypeptides disclosed in FIGS. 1 and 3,respectively, (i.e., UNQ516 and UNQ561), are shown to begin with amethionine residue designated as amino acid position 1, it isconceivable and possible that another methionine residue located eitherupstream or downstream from amino acid position 1 in FIG. 1 or FIG. 3may be employed as the starting amino acid residue.

The term “UNQ516” or “UNQ561” refer to the specific native sequencePRO1031 or PRO1122 protein, respectively, depicted in FIG. 1 or 3,respectively. Optionally, the PRO1031 or PRO1122 polypeptide is obtainedor obtainable by expressing the polypeptide encoded by the cDNA insertof the vector DNA59294-1381 or DNA62377-1381-1, under ATCC depositnumber 209866 or 203552, respectively.

“PRO1031 variant” or “PRO1122 variant” means an “active” PRO1031polypeptide or PRO1122 polypeptide, respectively, as defined belowhaving at least about 80% amino acid sequence identity with the PRO1031polypeptide or PRO1122 polypeptide, respectively, having the deducedamino acid sequence of residues (1) 1 or about 21 to about 180 shown inFIG. 1 (SEQ ID NO:1), or (2) 1 or about 19 to 197 shown in FIG. 3 (SEQID NO:3), respectively, for a full-length or mature native sequencePRO1031 or PRO1122 polypeptide, respectively. Such PRO1031 or PRO1122polypeptide variants include, for instance, PRO1031 polypeptides orPRO1122 polypeptides, respectively, wherein one or more amino acidresidues are added, substituted or deleted, at the N- or C-terminus orwithin the sequence of FIG. 1 (SEQ ID NO:1) or FIG. 3 (SEQ ID NO:3),respectively. Ordinarily, a PRO1031 or PRO1122 polypeptide variant willhave at least about 80% amino acid sequence identity, preferably atleast about 81% amino acid sequence identity, more preferably at leastabout 82% amino acid sequence identity, yet more preferably at leastabout 83% sequence identity, yet more preferably at least about 84%sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 96% sequence identity, yet morepreferably at least about 97% sequence identity, yet more preferably atleast about 98% sequence identity, yet more preferably at least about99% amino acid sequence identity with the amino acid sequence of FIG. 1(SEQ ID NO:1) or FIG. 3 (SEQ ID NO:3), with or without the signalpeptide (E.g., with signal peptide amino acid residues 1 to 180 of SEQID NO:1, 1 to 197 of SEQ ID NO:3, without signal peptide about 21 to 180of SEQ ID NO:1, about 19 to 197 of SEQ ID NO:3). The variants providedherein exclude native sequence PRO1031 and PRO1122 sequences as well thepolypeptides and nucleic acids described herein with which the PRO1031and PRO1122 polypeptides share 100% identity and/or which are alreadyknown in the art.

“Percent (%) amino acid sequence identity” with respect to the PRO1031amino acid sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in a PRO1031 polypeptide sequence, after aligningthe sequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as ALIGN, ALIGN-2, Megalign(DNASTAR) or BLAST (e.g., Blast, Blast-2, WU-Blast-2) software. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full length of the sequences being compared. For example, the %identity values used herein are generated using WU-BLAST-2 (Altschul etal., Methods in Enzymology 266: 460-480 (1996). Most of the WU-BLAST-2search parameters are set to the default values. Those not set todefault values, i.e., the adjustable parameters, are set with thefollowing values: overlap span=1, overlap fraction=0.125, word threshold(T)=11, and scoring matrix=BLOSUM 62. For purposes herein, a % aminoacid sequence identity value is determined by divided (a) the number ofmatching identical amino acid residues between the amino acid sequenceof the PRO1031 or PRO1122 polypeptide of interest and the comparisonamino acid sequence of interest (i.e., the sequence against which thePRO1031 or PRO1122 polypeptide of interest is being compared) asdetermined by WU-BLAST-2 by (b) the total number of amino acid residuesof the PRO1031 or PRO1122 polypeptide of interest, respectively.

A “PRO1031 or PRO1122 variant polynucleotide” or PRO1031 or PRO1122variant nucleic acid sequence” means an active PRO1031 or PRO1122polypeptide-encoding nucleic acid molecule as defined below having atleast about 65% nucleic acid sequence identity with the nucleotide acidsequence of nucleotides: (1) about 42 or about 102 to about 589 or about687 of the PRO1031-encoding nucleotide sequence shown in FIG. 2 (SEQ IDNO:2); or (2) about 59 or about 104 to about 640 or about 1043 of thePRO1122-encoding nucleotide sequence shown in FIG. 4 (SEQ ID NO:4),respectively. Ordinarily, a PRO1031 or PRO1122 polypeptide will have atleast about 65% nucleic acid sequence identity, more preferably at leastabout 70% nucleic acid sequence identity, yet more preferably at leastabout 75% nucleic acid sequence identity, yet more preferably at leastabout 80% nucleic acid sequence identity, yet more preferably at leastabout 81% nucleic acid sequence identity, yet more preferably at leastabout 82% nucleic acid sequence identity, yet more preferably at leastabout 83% nucleic acid sequence identity, yet more preferably at leastabout 84% nucleic acid sequence identity, yet more preferably at leastabout 85% nucleic acid sequence identity, yet more preferably at leastabout 86% nucleic acid sequence identity, yet more preferably at leastabout 87% nucleic acid sequence identity, yet more preferably at leastabout 88% nucleic acid sequence identity, yet more preferably at leastabout 89% nucleic acid sequence identity, yet more preferably at leastabout 90% nucleic acid sequence identity, yet more preferably at leastabout 91% nucleic acid sequence identity, yet more preferably at leastabout 92% nucleic acid sequence identity, yet more preferably at leastabout 93% nucleic acid sequence identity, yet more preferably at leastabout 94% nucleic acid sequence identity, yet more preferably at leastabout 95% nucleic acid sequence identity, yet more preferably at leastabout 96% nucleic acid sequence identity, yet more preferably at leastabout 97% nucleic acid sequence identity, yet more preferably at leastabout 98% nucleic acid sequence identity, yet more preferably at leastabout 99% nucleic acid sequence identity with the nucleic acid sequenceof nucleotides: 1) about 42 or about 102 to about 589 of thePRO1031-encoding nucleotide sequence shown in FIG. 2 (SEQ ID NO:2); or(2) about 59 or about 104 to about 640 of the PRO1122-encodingnucleotide sequence shown in FIG. 4 (SEQ ID NO:4), respectively.Variants specifically exclude or do not encompass the native nucleotidesequence, as well as those prior art sequences which share 100% identitywith the nucleotide sequences of the invention.

“Percent (%) nucleic acid sequence identity” with respect to the PRO1031or PRO1122 sequences identified herein is defined as the percentage ofnucleotides in a candidate sequence that are identical with thenucleotides in the PRO1031 sequence or PRO1122 sequence, respectively,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as ALIGN, Align-2, Megalign(DNASTAR), or BLAST (e.g., Blast, Blast-2) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull length of the sequences being compared. For purposes herein,however, % nucleic acid identity values are generated using theWU-BLAST-2 (BlastN module) computer program (Altschul et al., Methods inEnzymology 266: 460-480 (1996). Most of the WU-BLAST-2 search parametersare set to the default values. Those not set default values, i.e., theadjustable parameters, are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11 and scoringmatrix=BLOSUM62. For purposes herein, a % nucleic acid sequence identityvalue is determined by dividing (a) the number of matching identicalnucleotides between the nucleic acid sequence of the PROpolypeptide-encoding nucleic acid molecule of interest and thecomparison nucleic acid molecule of interest (i.e., the sequence againstwhich the PRO polypeptide-encoding nucleic acid molecule of interest isbeing compared) as determined by WU-BLAST-2 by (b) the total number ofnucleotides of the PRO polypeptide-encoding nucleic acid molecule ofinterest.

In other embodiments, the PRO1031 or PRO1122 variant polypeptides arenucleic acid molecules that encode an active PRO1031 or PRO1122polypeptide and which are capable of hybridizing, preferably understringent hybridization and wash conditions, to nucleotide sequencesencoding the full-length PRO1031 or PRO1122 polypeptide shown in FIG. 2(SEQ ID NO:2) or FIG. 4 (SEQ ID NO:4), respectively. This scope ofvariant polynucleotides specifically excludes those sequences which areknown as of the filing and/or priority dates of the present application.Furthermore, PRO1031 or PRO1122 variant polypeptides may also be thosethat are encoded by a PRO1031 or PRO1122 variant polynucleotide,respectively.

The term “positives”, in the context of sequence comparison performed asdescribed above, includes residues in the sequences compared that arenot identical but have similar properties (e.g., as a result ofconservative substitutions). The % identity value of positives isdetermined by the fraction of residues scoring a positive value in theBLOSUM 62 matrix. This value is determined by dividing (a) the number ofamino acid residues scoring a positive value in the BLOSUM62 matrix ofWU-BLAST-2 between the PRO1031 or PRO1122 polypeptide amino acidsequence of interest and the comparison amino acid sequence (i.e., theamino acid sequence against which the PRO1031 or PRO1122 polypeptidesequence is being compared) as determined by WU-BLAST-2 by (b) the totalnumber of amino acid residues of the PRO1031 or PRO1122 polypeptide ofinterest.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Preferably, theisolated polypeptide is free of association with all components withwhich it is naturally associated. Contaminant components of its naturalenvironment are materials that would typically interfere with diagnosticor therapeutic uses for the polypeptide, and may include enzymes,hormones, and other proteinaceous or non-proteinaceous solutes. Inpreferred embodiments, the polypeptide will be purified (1) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (2) tohomogeneity by SDS-PAGE under non-reducing or reducing conditions usingCoomassie blue or, preferably, silver stain. Isolated polypeptideincludes polypeptide in situ within recombinant cells, since at leastone component of the PRO1031 or PRO1122 polypeptide natural environmentwill not be present. Ordinarily, however, isolated polypeptide will beprepared by at least one purification step.

An “isolated” PRO1031 or PRO1122 polypeptide-encoding nucleic acidmolecule is a nucleic acid molecule that is identified and separatedfrom at least one contaminant nucleic acid molecule with which it isordinarily associated in the natural source of the PRO1031 polypeptide-or PRO1122 polypeptide-encoding nucleic acid. An isolated PRO1031polypeptide- or PRO1122 polypeptide-encoding nucleic acid molecule isother than in the form or setting in which it is found in nature.Isolated PRO1031 polypeptide- or PRO1122 polypeptide-encoding nucleicacid molecules therefore are distinguished from the PRO1031 polypeptide-or PRO1122 polypeptide-, respectively, encoding nucleic acid molecule asit exists in natural cells. However, an isolated PRO1031 polypeptide- orPRO1122 polypeptide-encoding nucleic acid molecule includes PRO1031polypeptide- or PRO1122 polypeptide-, respectively, encoding nucleicacid molecules contained in cells that ordinarily express PRO1031polypeptide or PRO1122 polypeptide, where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes required higher temperaturesfor proper annealing, while short probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reactions more stringent,while lower temperatures less so. For additional details and explanationof stringency of hybridization reactions, see Ausubel et al., CurrentProtocols in Molecular Biology, Wiley Interscience Publishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that” (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning. A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” where used herein refers to a chimericpolypeptide comprising a PRO1031 or PRO1122 polypeptide, or domainsequence thereof, fused to a “tag polypeptide”. The tag polypeptide hasenough residues to provide an epitope against which an antibody may bemade, or which can be identified by some other agent, yet is shortenough such that it does not interfere with the activity of the PRO1031or PRO1122 polypeptide. The tag polypeptide preferably is also fairlyunique so that the antibody does not substantially cross-react withother epitopes. Suitable tag polypeptides generally have at least sixamino acid residues and usually between about 8 to about 50 amino acidresidues (preferably, between about 10 to about 20 residues).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesion”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3 or IgG-4 subtypes, IgA (including IgG-1 and IgA-2), IgE,IgD or IgM.

The term “antibody” is used in the broadest sense and specificallycovers single anti-PRO1031 or anti-PRO1122 polypeptide monoclonalantibodies (including agonist, antagonist, and neutralizing antibodies),anti-PRO1031 or anti-PRO1122, respectively, antibody compositions withpolyepitopic specificity, single-chain anti-PRO1031 or anti-PRO1122antibodies, and fragments of anti-PRO1031 or anti-PRO1122 antibodies.The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts.

“Active” or “activity” for the purposes herein refers to form(s) ofPRO1031 or PRO1122 which retain the biologic and/or immunologicactivities of native or naturally-occurring PRO1031 or PRO1122,respectively, polypeptide. Elaborating further, “biological” activityrefers to a biological function (either inhibitory or stimulatory)caused by a native or naturally-occurring PRO1031 or PRO1122 other thanthe ability to induce the production of an antibody against an antigenicepitope possessed by a native or naturally-occurring PRO1031 or PRO1122and an “immunological” activity refers only to the ability to induce theproduction of an antibody against an antigenic epitope possessed by anative or naturally-occurring PRO1031 or PRO1122. A preferred biologicalactivity includes, for example, the release of TNF-α from THP1 cells. Analternative activity is the reduction in IL-1α induced NO (nitric oxide)production from articular cartilage.

“Degenerative cartilagenous disorder” describes a host of disorders thatis characterized principally by the destruction of the cartilage matrix.Additional pathologies includes nitric oxide production, and elevatedproteoglycan breakdown. Exemplary disorders encompassed within thisdefinition, include, for example, arthritis (e.g., osteoarthritis,rheumatoid arthritis, psoriatic arthritis), sepsis, ulcerative colitis,psoriasis, multiple sclerosis, type I diabetes, giant cell arthritis,systemic lupus erythematosus and Sjögren's syndrome.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native PRO1031 or PRO1122 polypeptide disclosedherein. In a similar manner, the term “agonist” is used in the broadestsense and includes any molecule that mimics a biological activity of anative PRO1031 or PRO1122 polypeptide disclosed herein. Suitable agonistor antagonist molecules specifically include agonist or antagonistantibodies or antibody fragments, fragments or amino acid sequencevariants of native PRO1031 or PRO1122 polypeptides, peptides, smallorganic molecules, etc. Method for identifying agonists or antagonistsof a PRO1031 or PRO1122 polypeptide may comprise contacting a PRO1031 orPRO1122 polypeptide with a candidate agonist or antagonist molecule andmeasuring a detectable change in one or more biological activitiesnormally associated with the PRO1031 or PRO1122 polypeptide.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.The term “antibody” is used in the broadest sense and specificallycovers, without limitation, intact monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g. bispecific antibodies) formedfrom at least two intact antibodies, and antibody fragments so long asthey exhibit the desired biological activity.

The terms “treating”, “treatment” and “therapy” as used herein refer tocurative therapy, prophylactic therapy, and preventative therapy. Anexample of “preventative therapy” is the prevention or lessened targetedpathological condition or disorder. Those in need of treatment includethose already with the disorder as well as those prone to have thedisorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

The term “mammal” as used herein refers to any mammal classified as amammal, including humans, domestic and farm animals, and zoo, sports orpet animals, such as cattle (e.g. cows), horses, dogs, sheep, pigs,rabbits, goats, cats, etc. In a preferred embodiment of the invention,the mammal is a human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

A “therapeutically-effective amount” is the minimal amount of activeagent (e.g., PRO1031, PRO1122, antagonist or agonist thereof) which isnecessary to impart therapeutic benefit to a mammal. For example a“therapeutically-effective amount” to a mammal suffering or prone tosuffering or to prevent it from suffering from a degenerativecartilagenous disorder is such an amount which induces, ameliorates orotherwise causes an improvement in the pathological symptoms, diseaseprogression, physiological conditions associated with or resistance tosuccumbing to a disorder principally characterized by the destruction ofthe cartilage matrix.

“Carriers” as used herein include pharmaceutically-acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically-acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecule weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN®, polyethylene glycol (PEG), and PLURONICS™.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies (Zapata et al., ProteinEngin. 8(10): 1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDR specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fv fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA and IgA2.

“Single-chain Fv” or “sFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domain which enables the sFv toform the desired structure for antigen binding. For a review of sFv, seePluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097, WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA 90: 6444-6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue, or preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself (e.g., radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alternation of a substratecompound or composition which is detectable.

“Solid phase” is meant to be a non-aqueous matrix to which the antibodyof the present invention can adhere. Examples of solid phasesencompassed herein include those formed partially or entirely of glass(e.g., controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromotagraphy column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a PRO1031 or PRO1122 polypeptide or antibody thereto) to amammal. The components of the liposome are commonly arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes.

A “small molecule” is defined herein to have a molecule weight belowabout 500 Daltons.

The term “modulate” means to affect (e.g., either upregulate,downregulate or otherwise control) the level of a signaling pathway.Cellular processes under the control of signal transduction include, butare not limited to, transcription of specific genes, normal cellularfunctions, such as metabolism, proliferation, differentiation, adhesion,apoptosis and survival, as well as abnormal processes, such astransformation, blocking of differentiation and metastasis.

II. Compositions and Methods of the Invention

A. Full-Length PRO1031 or PRO1122 Polypeptide

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO1031 or PRO1122. In particular, Applicants have identified andisolated cDNA encoding a PRO1031 (e.g., UNQ516, IL-17B, SEQ ID NO:1) andPRO1122 (e.g., UNQ561, IL-17C, SEQ ID NO:3) polypeptide, as disclosed infurther detail in the Examples below. Using BLAST and FastA sequencealignment computer programs, Applicants found that various portions ofthe PRO1031 and PRO1122 polypeptide have sequence identity with IL-17.Accordingly, it is presently believed that PRO1031 and PRO1122polypeptide disclosed in the present application are newly identifiedmembers of the cytokine family and thus may be involved in inflammationand/or the immune system function.

As presented earlier, the term “PRO1031” or “PRO1122” refers to thenative sequence and variants, whereas the terms “UNQ516” or “UNQ561”refer to the specific amino acid sequences of FIG. 1 (SEQ ID NO:1) andFIG. 3 (SEQ ID NO:3), respectively, and/or the proteins encoded by thecDNA deposited with the American Type Culture Collection, under Depositnumbers 209866 and 203552, respectively.

As disclosed in the Examples below, cDNA clone designated herein asDNA59294-1381 and DNA62377-1381-1 have been deposited with the ATCC. Theactual nucleotide sequence of the clone can be readily determined by theskilled artisan by sequencing of the deposited clone using routinemethods in the art. The predicted amino acid sequence can be determinedfrom the nucleotide sequence using routine skill. For the PRO1031 orPRO1122 polypeptide and encoding nucleic acid described herein,Applicants have identified what is believed to be the reading frame bestidentifiable with the sequence information available at the time.

B. PRO1031 and PRO1122 Variants

In addition to the full-length native sequence PRO1031 or PRO1122polypeptide described herein, it is contemplated that PRO1031 or PRO1122variants can be prepared. PRO1031 or PRO1122 variants can be prepared byintroducing appropriate nucleotide changes into the PRO1031- orPRO1122-encoding DNA, or by synthesis of the desired PRO1031 or PRO1122polypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the PRO1031 or PRO1122polypeptide, such as changing the number or position of glycosylationsites or altering the membrane anchoring characteristics.

Variations in the native full-length sequence PRO1031 or PRO1122 or invarious domains of the PRO1031 or PRO1122 polypeptide described herein,can be made, for example, using any of the techniques and guidelines forconservative and non-conservative mutations set forth, for instance, inU.S. Pat. No. 5,364,934. Variations may be a substitution, deletion orinsertion of one or more codons encoding the PRO1031 or PRO1122polypeptide that results in a change in the amino acid sequence of thePRO1031 or PRO1122 polypeptide as compared with the native sequencePRO1031 or PRO1122. Optionally the variation is by substitution of atleast one amino acid with any other amino acid in one or more of thedomains of the PRO1031 or PRO1122 polypeptide. Guidance in determiningwhich amino acid residue may be inserted, substituted or deleted withoutadversely affecting the desired activity may be found by comparing thesequence of the PRO1031 or PRO1122 polypeptide with that of homologousknown protein molecules and minimizing the number of amino acid sequencechanges made in regions of high homology. Amino acid substitutions canbe the result of replacing one amino acid with another amino acid havingsimilar structural and/or chemical properties, such as the replacementof a leucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of 1 to 5 aminoacids. The variation allowed may be determined by systematically makinginsertions, deletions or substitutions of amino acids in the sequenceand testing the resulting variants for activity (such as in any of thein vitro assays described in the Examples below) for activity exhibitedby the full-length or mature native sequence.

PRO1031 or PRO1122 polypeptide fragments are provided herein. Suchfragments may be truncated at the N-terminus or C-terminus, or may lackinternal residues, for example, when compared with a full length ornative protein. Certain fragments lack amino acid residues that are notessential for a desired biological activity of the PRO1031 or PRO1122polypeptide.

PRO1031 or PRO1122 fragments may be prepared by any of a number ofconventional techniques. Desired peptide fragments may be chemicallysynthesized. An alternative approach involves generating PRO1031 orPRO1122 fragments by enzymatic digestion, e.g., by treating the proteinwith an enzyme known to cleave proteins at sites defined by particularamino aid residues, or by digesting the DNA with suitable restrictionenzymes and isolating the desired fragment. Yet another suitabletechnique involves isolating and amplifying a DNA fragment encoding adesired polypeptide fragment, by polymerase chain reaction (PCR).Oligonucleotides that define the desired termini of the DNA fragment areemployed at the 5′ and 3′ primers in the PCR. Preferably, PRO1031 orPRO1122 polypeptide fragments share at least one biological and/orimmunological activity with the native PRO1031 or PRO1122 polypeptideshown in FIG. 1 (SEQ ID NO:1) or FIG. 3 (SEQ ID NO:3).

In particular embodiments, conservative substitutions of interest areshown in Table 1 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 1, oras further described below in reference to amino acid classes, areintroduced and the products screened. TABLE 1 Conservative SubstitutionsPreferred Original residue Example substitutions substitutions Ala (A)val, leu, ile val Arg (R) lys, gln, asn lys Asn (N) gln, his, lys, arggln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly(G) pro, ala ala His (H) asn, gln, lys, arg arg Ile (I) leu, val, met,ala, phe, leu norleucine Leu (L) norleucine, ile, val, met, ala, ile pheLys (K) arg, gln, asn arg Met (M) leu, phe, ile leu Phe (F) leu, val,ile, ala, tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp(W) tyr, phe tyr Tyr (Y) trp, phe, thr, ser phe Val (V) ile, leu, met,phe, ala, leu norleucine

Substantial modifications in function or immunological identity of thePRO1031 or PRO1122 polypeptide are accomplished by selectingsubstitutions that differ significantly in their effect on maintaining(a) the structure of the polypeptide backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site, or (c) thebulk of the side chain. Naturally occurring residues are divided intogroups based on common side-chain properties:

(1) hydrophobic: sys, ser, thr;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites, or more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO1031-encoding variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant. Alanine is alsotypically preferred because it is the most common amino acid. Further,it is frequently found in both buried and exposed positions [Creighton,The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1(1976)]. If alanine substitution does not yield adequate amounts ofvariant, an isoteric amino acid can be used.

C. Modifications of PRO1031 or PRO1122

Covalent modifications of PRO1031 or PRO1122 polypeptides are includedwithin the scope of this invention. One type of covalent modificationincludes reacting targeted amino acid residues of a PRO1031 or PRO1122polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues of aPRO1031 or PRO1122 polypeptide. Derivatization with bifunctional agentsis useful, for instance, for crosslinking PRO1031 or PRO1122 to awater-insoluble support matrix or surface for use in the method forpurifying anti-PRO1031 or PRO1122 antibodies, and vice-versa. Commonlyused crosslinking agents include, e.g.,1,1-bis(diazo-acetyl)-2-phenylethane, glutaraldehyde,N-hydroxy-succinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis-(succinimidylproprionate), bifunctional maleimidessuch as bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)-dithio]proprioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the PRO1031 or PRO1122polypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of the polypeptide. “Alteringthe native glycosylation pattern” is intended for purposes herein tomean deleting one or more carbohydrate moieties found in native sequencePRO1031 or PRO1122 polypeptide, and/or adding one or more glycosylationsites that are not present in the native sequence PRO1031 or PRO1122polypeptide. Additionally, the phrase includes qualitative changes inthe glycosylation of the native proteins, involving a change in thenature and proportions of the various carbohydrate moieties present.

Addition of glycosylation sites to PRO1031 or PRO1122 polypeptides maybe accomplished by altering the amino acid sequence thereof. Thealteration may be made, for example, by the addition of, or substitutionby, one or more serine or threonine residues to the native sequencePRO1031 or PRO1122 polypeptide (for O-linked glycosylation sites). ThePRO1031 or PRO1122 amino acid sequence may optionally be altered throughchanges at the DNA level, particularly by mutating the DNA encoding thePRO1031 or PRO1122 polypeptide at preselected bases such that codons aregenerated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thePRO1031 or PRO1122 polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Such methods are described in the art,e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin and Wriston,CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the PRO1031 or PRO1122polypeptide may be accomplished chemically or enzymatically or bymutational substitution of codons encoding for amino acid residues thatserve as targets for glycosylation. Chemical deglycosylation techniquesare known in the art and described, for instance, by Hakimuddin, et al.,Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal.Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Another type of covalent modification of PRO1031 or PRO1122 compriseslinking the PRO1031 or PRO1122 polypeptide, respectively, to one of avariety of nonproteinaceous polymers, e.g., polyethylene glycol,polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

PRO1031 or PRO1122 polypeptides of the present invention may also bemodified in a way to form chimeric molecules comprising a PRO1031 orPRO1122 polypeptide, respectively, fused to another, heterologouspolypeptide or amino acid sequence. In one embodiment, such a chimericmolecule comprises a fusion of a PRO1031 or PRO1122 polypeptide with atag polypeptide which provides an epitope to which an anti-tag antibodycan selectively bind. The epitope tag is generally placed at the amino-or carboxyl-terminus of the PRO1031 or PRO1122 polypeptide. The presenceof such epitope-tagged forms of a PRO1031 or PRO1122 polypeptide can bedetected using an antibody against the tag polypeptide. Also, provisionof the epitope tag enables the PRO1031 or PRO1122 polypeptide to bereadily purified by affinity purification using an anti-tag antibody oranother type of affinity matrix that binds to the epitope tag.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of a PRO1031 or PRO1122 polypeptide with an immunoglobulin or aparticular region of an immunoglobulin. For a bivalent form of thechimeric molecule, such a fusion could be to the Fc region of an IgGmolecule. The Ig fusions preferably include the substitution of asoluble transmembrane domain deleted or inactivated) form of a PRO1031or PRO1122 polypeptide in place of at least one variable region withinan Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130, issued Jun. 27,1995.

In yet a further embodiment, the PRO1031 or PRO1122 polypeptides of thepresent invention may also be modified in a way to form a chimericmolecule comprising a PRO1031 or PRO1122 polypeptide fused to a leucinezipper. Various leucine zipper polypeptides have been described in theart. See, e.g., Landschulz et al., Science 240:1759 (1988); WO 94/10308;Hoppe et al., FEBS Letters 344:1991 (1994); Maniatis et al., Nature341:24 (1989). It is believed that use of a leucine zipper fused to aPRO1031 or PRO1122 polypeptide may be desirable to assist in dimerizingor trimerizing soluble PRO1031 or PRO1122 polypeptide in solution. Thoseskilled in the art will appreciate that the leucine zipper may be fusedat either the N- or C-terminal end of the PRO1031 or PRO1122 molecule.

D. Preparation of PRO1031 or PRO1122

The description below relates primarily to production of PRO1031 orPRO1122 by culturing cells transformed or transfected with a vectorcontaining PRO1031 or PRO1122 polypeptide encoding nucleic acid. It is,of course, contemplated that alternative methods, which are well knownin the art, may be employed to prepare PRO1031 or PRO1122 polypeptides.For instance, the PRO1031 or PRO1122 sequence, or portions thereof, maybe produced by direct peptide synthesis using solid-phase techniques[see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. FreemanCo., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc.,85:2149-2154 (1963)]. In vitro protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may beaccomplished, for instance, using an Applied Biosystems PeptideSynthesizer (Foster City, Calif.) using manufacturer's instructions.Various portions of PRO1031 or PRO1122 polypeptides may be chemicallysynthesized separately and combined using chemical or enzymatic methodsto produce a full-length PRO1031 or PRO1122 polypeptide.

1. Isolation of DNA Encoding PRO1031

DNA encoding a PRO1031 or PRO1122 polypeptide may be obtained from acDNA library prepared from tissue believed to possess the PRO1031 orPRO1122 mRNA and to express it at a detectable level. Accordingly, humanPRO1031- or PRO1122-encoding DNA can be conveniently obtained from acDNA library prepared from human tissue, such as described in theExamples. The PRO1031- or PRO1122-encoding gene may also be obtainedfrom a genomic library or by known synthetic procedures (e.g., automatedsynthetic procedures, oligonucleotide synthesis).

Libraries can be screened with probes (such as antibodies to a PRO1031or PRO1122 polypeptide or oligonucleotides of at least about 20-80bases) designed to identify the gene of interest or the protein encodedby it. Screening the cDNA or genomic library with the selected probe maybe conducted using standard procedures, such as described in Sambrook etal., Molecular Cloning. A Laboratory Manual (New York: Cold SpringHarbor Laboratory Press, 1989). An alternative means to isolate the geneencoding PRO1031 is to use PCR methodology [Sambrook et al., supra;Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring HarborLaboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein (e.g.,through sequence alignment using computer software programs such asALIGN, DNAstar, BLAST, BLAST-2, INHERIT and ALIGN-2 which employ variousalgorithms to measure homology).

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for PRO1031 or PRO1122 polypeptide productionand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. The culture conditions, such as media,temperature, pH and the like, can be selected by the skilled artisanwithout undue experimentation. In general, principles, protocols, andpractical techniques for maximizing the productivity of cell culturescan be found in Mammalian Cell Biotechnology: A Practical Approach, M.Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

Methods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Depending on the host cell used,transformation is performed using standard techniques appropriate tosuch cells. The calcium treatment employing calcium chloride, asdescribed in Sambrook et al., supra, or electroporation is generallyused for prokaryotes or other cells that contain substantial cell-wallbarriers. Infection with Agrobacterium tumefaciens is used fortransformation of certain plant cells, as described by Shaw et al.,Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the nucleic acid (e.g.,DNA) in the vectors herein include prokaryote, yeast, or highereukaryote cells. Suitable prokaryotes include but are not limited toeubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as E. coli. Various E. coli strains arepublicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E.coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5772(ATCC 53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebisella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD266,710, published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA, ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 40B4, which isstrain 37D6 with a non-kanamycin resistant degP deletion mutation; andan E. coli strain having mutant periplasmic protease disclosed in U.S.Pat. No. 4,946,783 issued 7 Aug. 1990. Alternatively, in vivo methods ofcloning, e.g., PCR or other nucleic acid polymerase reactions, aresuitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for PRO1031- orPRO1122-encoding vectors. Saccharomyces cerevisiae is a commonly usedlower eukaryotic host microorganism. Others include Schizosaccharomycespombe (Beach and Nature, Nature 290: 140[1981]; EP 139,383 published 2May 1995); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9: 968-975 (1991) such as e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol. 737 [1983]), K.fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906); Vanden Berg et al., Bio/Technology 8: 135 (1990)); K. thermotolerans, andK. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Sreekrishna et al., J. basic Microbiol. 28: 265-278 [1988]); Candid;Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc.Natl. Acad. Sci. USA 76: 5359-5263 [1979]); Schwanniomyces such asSchwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990); andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357 published 10 Jan. 19910, and Aspergillus hosts such as A.nidulans (Balance et al., Biochem. Biophys. Res. Commun. 112: 284-289[1983]; Tilburn et al., Gene 26: 205-221 [1983]; Yelton et al., Proc.Natl. Acad. Sci. USA 81: 1470-1474 [1984]) and A. niger (Kelly andHynes, EMBO J. 4: 475-479 [1985]). Methylotropic yeasts are selectedfrom the genera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeast may be found in C. Antony, TheBiochemistry of Methylotrophs 269 (1982).

Suitable host cells for the expression of glycosylated PRO1031 orPRO1122 are derived from multicellular organisms. Examples ofinvertebrate cells include insect cells such as Drosophila S2 andSpodoptera Sf9, Spodoptera high5 as well as plant cells. Examples ofuseful mammalian host cell lines include Chinese hamster ovary (CHO) andCOS cells. More specific examples include monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol., 36:59 (1977)); Chinese hamster ovarycells/-DHFR(CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251(1980)); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51). Theselection of the appropriate host cell is deemed to be within the skillin the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding the desiredPRO1031 or PRO1122 polypeptide may be inserted into a replicable vectorfor cloning (amplification of the DNA) or for expression. Variousvectors are publicly available. The vector may, for example, be in theform of a plasmid, cosmid, viral particle, or phage. The appropriatenucleic acid sequence may be inserted into the vector by a variety ofprocedures. In general, DNA is inserted into an appropriate restrictionendonuclease site(s) using techniques known in the art. Vectorcomponents generally include, but are not limited to, one or more of asignal sequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.Construction of suitable vectors containing one or more of thesecomponents employs standard ligation techniques which are known to theskilled artisan.

The PRO1031 or PRO1122 polypeptide may be produced recombinantly notonly directly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence or other polypeptide havinga specific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the PRO1031- or PRO1122-encoding DNA thatis inserted into the vector. The signal sequence may be a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces α-factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression, mammaliansignal sequences may be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up thePRO1031-or PRO1122-encoding nucleic acid, such as DHFR or thymidinekinase. An appropriate host cell when wild-type DHFR is employed is theCHO cell line deficient in DHFR activity, prepared and propagated asdescribed by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980).A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979);Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157(1980)]. The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the PRO1031- or PRO1122-encoding nucleic acid sequence todirect mRNA synthesis. Promoters recognized by a variety of potentialhost cells are well known. Promoters suitable for use with prokaryotichosts include the β-lactamase and lactose promoter systems [Chang etal., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters suchas the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25(1983)]. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding thePRO1031 or PRO1122 polypeptide.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

PRO1031 or PRO1122 transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g.,the actin promoter or an immunoglobulin promoter, and from heat-shockpromoters, provided such promoters are compatible with the host cellsystems.

Transcription of a DNA encoding a PRO1031 or PRO1122 polypeptide byhigher eukaryotes may be increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp, that act on a promoter to increase its transcription.Many enhancer sequences are now known from mammalian genes (globin,elastase, albumin, α-fetoprotein, and insulin). Typically, however, onewill use an enhancer from a eukaryotic cell virus. Examples include theSV40 enhancer on the late side of the replication origin (bp 100-270),the cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO1031 or PRO1122 coding sequence, but is preferably located at a site5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding PRO1031 or PRO1122.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of PRO1031 or PRO1122 polypeptides in recombinantvertebrate cell culture are described in Gething et al., Nature,293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060;and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO1031 or PRO1122 polypeptide or against a synthetic peptide based onthe DNA sequences provided herein or against exogenous sequence fused toPRO1031- or PRO1122-encoding DNA and encoding a specific antibodyepitope.

5. Purification of Polypeptide

Forms of PRO1031 or PRO1122 may be recovered from culture medium or fromhost cell lysates. If membrane-bound, it can be released from themembrane using a suitable detergent solution (e.g. Triton-X 100) or byenzymatic cleavage. Cells employed in expression of PRO1031 or PRO1122polypeptides can be disrupted by various physical or chemical means,such as freeze-thaw cycling, sonication, mechanical disruption, or celllysing agents.

It may be desired to purify PRO1031 or PRO1122 from recombinant cellproteins or polypeptides. The following procedures are exemplary ofsuitable purification procedures: by fractionation on an ion-exchangecolumn; ethanol precipitation; reverse phase HPLC; chromatography onsilica or on a cation-exchange resin such as DEAE; chromatofocusing;SDS-PAGE; ammonium sulfate precipitation; gel filtration using, forexample, Sephadex G-75; protein A Sepharose columns to removecontaminants such as IgG; and metal chelating columns to bindepitope-tagged forms of the PRO1031 or PRO1122 polypeptide. Variousmethods of protein purification may be employed and such methods areknown in the art and described for example in Deutscher, Methods inEnzymology, 182 (1990); Scopes, Protein Purification: Principles andPractice, Springer-Verlag, New York (1982). The purification step(s)selected will depend, for example, on the nature of the productionprocess used and the particular PRO1031 or PRO1122 polypeptide produced.

E. Uses for PRO1031

Nucleotide sequences (or their complement) encoding PRO1031 or PRO1122polypeptides have various applications in the art of molecular biology,including uses as hybridization probes, in chromosome and gene mappingand in the generation of anti-sense RNA and DNA. PRO1031- orPRO1122-encoding nucleic acid will also be useful for the preparation ofPRO1031 or PRO1122 polypeptides by the recombinant techniques describedherein.

The full-length DNA59294-1381 nucleotide sequence (SEQ ID NO:2),full-length DNA62377-1381-1 nucleotide sequence (SEQ ID NO:4) or thefull-length native sequence PRO1031 or PRO1122 nucleotide-encodingsequence, or portions thereof, may be used as hybridization probes for acDNA library to isolate the full-length PRO1031 or PRO1122 gene or toisolate still other genes (for instance, those encodingnaturally-occurring variants of PRO1031, PRO1122 or the same from otherspecies) which have a desired sequence identity to the PRO1031 orPRO1122 nucleotide sequence disclosed in FIG. 2 (SEQ ID NO:2) or FIG. 4(SEQ ID NO:4), respectively. Optionally, the length of the probes willbe about 20 to about 50 bases. The hybridization probes may be derivedfrom the DNA59294-1381 or DNA62377-1381-1 nucleotide sequence of SEQ IDNO:2 or SEQ ID NO:4, respectively, as shown in FIG. 2 or FIG. 4,respectively, or from genomic sequences including promoters, enhancerelements and introns of native sequence PRO1031- or PRO1122-encodingDNA. By way of example, a screening method will comprise isolating thecoding region of the PRO1031 or PRO1122 gene using the known DNAsequence to synthesize a selected probe of about 40 bases. Hybridizationprobes may be labeled by a variety of labels, including radionucleotidessuch as ³²P or ³⁵S, or enzymatic labels such as alkaline phosphatasecoupled to the probe via avidin/biotin coupling systems. Labeled probeshaving a sequence complementary to that of the PRO1031 or PRO1122 geneof the present invention can be used to screen libraries of human cDNA,genomic DNA or mRNA to determine which members of such libraries theprobe hybridizes to. Hybridization techniques are described in furtherdetail in the Examples below.

Any EST sequence (or fragment thereof) disclosed in the presentapplication may similarly be employed as probes, using the methodsdisclosed herein.

Other useful fragments of the PRO1031 or PRO1122 nucleic acids includeantisense or sense oligonucleotides comprising a single-stranded nucleicacid sequence (either RNA or DNA) capable of binding to target PRO1031or PRO1122 mRNA (sense) of PRO1031 or PRO1122 DNA (anti-sense)sequences. Antisense or sense oligonucleotides, according to the presentinvention, comprise a fragment of the coding region of PRO1031 orPRO1122 DNA. Such a fragment generally comprises at least about 14nucleotides, preferably from about 14 to 30 nucleotides. The ability toderive an antisense or a sense oligonucleotide, based upon a cDNAsequence encoding a given protein is described in, for example, Steinand Cohen, Cancer Res. 48:2659 (1988) and van der Krol et al.,BioTechniques 6: 958 (1988).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of PRO1031 orPRO1122 proteins. Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO 91/06629) andwherein such sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10048, and other moieties that increaseaffinity of the oligonucleotide for a target nucleic acid sequence, suchpoly-L-lysine. Further still, intercalating agents, such as ellipticine,and alkylating agents or metal complexes may be attached to sense orantisense oligonucleotides to modify binding specificities of theantisense or sense oligonucleotide for the target nucleotide sequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated CDT5A, DCT5B and DCT5C (see WO 90/13641).

Sense of antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related PRO1031 or PRO1122sequences.

Nucleotide sequences encoding a PRO1031 or PRO1122 polypeptide can alsobe used to construct hybridization probes for mapping the gene whichencodes that PRO1031 or PRO1122 polypeptide and for the genetic analysisof individuals with genetic disorders. The nucleotide sequences providedherein may be mapped to a chromosome and specific regions of achromosome using known techniques, such as in situ hybridization,linkage analysis against known chromosomal markers, and hybridizationscreening with libraries.

When the coding sequences for PRO1031 or PRO1122 encode a protein whichbinds to another protein (example, where the PRO1031 or PRO1122polypeptide, respectively, functions as a receptor), the PRO1031 orPRO1122 polypeptide, respectively, can be used in assays to identify theother proteins or molecules involved in the binding interaction. By suchmethods, inhibitors of the receptor/ligand binding interaction can beidentified. Proteins involved in such binding interactions can also beused to screen for peptide or small molecule inhibitors or agonists ofthe binding interaction. Also, the receptor PRO1031 or PRO1122polypeptide can be used to isolate correlative ligand(s). Screeningassays can be designed to find lead compounds that mimic the biologicalactivity of a native PRO1031 or PRO1122 or a receptor for PRO1031 orPRO1122, respectively. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.Small molecules contemplated include synthetic organic or inorganiccompounds. The assays can be performed in a variety of formats,including protein-protein binding assays, biochemical screening assays,immunoassays and cell based assays, which are well characterized in theart.

Nucleic acids which encode PRO1031 or PRO1122 polypeptide or any of itsmodified forms can also be used to generate either transgenic animals or“knock out” animals which, in turn, are useful in the development andscreening of therapeutically useful reagents. A transgenic animal (e.g.,a mouse or rat) is an animal having cells that contain a transgene,which transgene was introduced into the animal or an ancestor of theanimal at a prenatal, e.g., an embryonic stage. A transgene is a DNAwhich is integrated into the genome of a cell from which a transgenicanimal develops. In one embodiment, cDNA encoding PRO1031 or PRO1122polypeptide can be used to clone genomic DNA encoding PRO1031 or PRO1122in accordance with established techniques and the genomic sequences usedto generate transgenic animals that contain cells which express DNAencoding PRO1031 or PRO1122. Methods for generating transgenic animals,particularly animals such as mice or rats, have become conventional inthe art and are described, for example, in U.S. Pat. Nos. 4,736,866 and4,870,009. Typically, particular cells would be targeted for PRO1031 orPRO1122 transgene incorporation with tissue-specific enhancers.Transgenic animals that include a copy of a transgene encoding PRO1031or PRO1122 introduced into the germ line of the animal at an embryonicstage can be used to examine the effect of increased expression of DNAencoding PRO1031 or PRO1122. Such animals can be used as tester animalsfor reagents thought to confer protection from, for example,pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of PRO1031 or PRO1122 can be used toconstruct a PRO1031 or PRO1122, respectively, “knock out” animal whichhas a defective or altered gene encoding PRO1031 or PRO1122,respectively, as a result of homologous recombination between theendogenous gene encoding PRO1031 or PRO1122, respectively, and alteredgenomic DNA encoding PRO1031 or PRO1122, respectively, introduced intoan embryonic cell of the animal. For example, cDNA encoding PRO1031 orPRO1122, respectively, can be used to clone genomic DNA encoding PRO1031or PRO1122, respectively, in accordance with established techniques. Aportion of the genomic DNA encoding PRO1031 or PRO1122, respectively,can be deleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69:915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells. A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-152]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the PRO1031 or PRO1122 polypeptide.

Nucleic acid encoding the PRO1031 or PRO1122 polypeptides may also beused in gene therapy. In gene therapy applications, gene are introducedinto cells in order to achieve in vivo synthesis of a therapeuticallyeffective genetic product, for example for replacment of a defectivegene. “Gene therapy” includes both conventional gene therapy where alasting effect is achieved by a single treatment, and the administrationof gene therapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where act asinhibitors, despite their low intracellular concentrations caused bytheir restricted uptake by the cell membrane. Zamecnik et al., Proc.Natl. Acad. Sci. USA 83: 4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g., by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cell in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11: 205-210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cells, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may by used for targeting and/or tofacilitate uptake, e.g., capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, protein that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example by Wu et al., J.Biol. Chem. 262: 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad.Sci. USA 87: 3410-3414 (1990). For a review of gene marking and genetherapy protocols see Anderson et al., Science 256: 808-813 (1992).

The PRO1031 or PRO1122 polypeptides described herein may also beemployed as molecular weight markers for protein electrophoresispurposes.

The nucleic acid molecule encoding the PRO1031 or PRO1122 polypeptidesor fragments thereof described herein are useful for chromosomeidentification. In this regard, there exists an ongoing need to identitynew chromosome markers, since relatively few chromosome markingreagents, based upon actual sequence data are presently available. EachPRO1031 or PRO1122 nucleic acid molecule of the present invention can beused as a chromosome marker.

The PRO1031 or PRO1122 polypeptides and nucleic acid molecules of thepresent invention may also be used for tissue typing, wherein thePRO1031 or PRO1122 polypeptides of the present invention may bedifferentially expressed in one tissue as compared to another. PRO1031or PRO112 nucleic acid molecules will find use for generating probes forPCR, Northern analysis, Southern analysis and Western analysis.

PRO1031 or PRO1122 polypeptides of the present invention which possessbiological activity related to that of IL-17 may be employed both invivo for therapeutic purposes and in vitro. Those of ordinary skill inthe art will well know how to employ the PRO1031 or PRO1122 polypeptidesof the present invention for such purposes.

PRO1031 or PRO1122 can be used in assays with the polypeptides to whichthey have identity with to determine the relative activities. Theresults can be applied accordingly.

An alignment of the predicted amino acid sequence of IL-17B (e.g.,UNQ516) (SEQ ID NO:1) and IL-17C (UNQ561) (SEQ ID NO:3) with the knownsequence of IL-17 (SEQ ID NO:11), show that this is a family of relatedsequences with a 26-28% amino acid identity between the three members(FIG. 7). All three polypeptides contain a hydrophobic sequence at theN-terminus that is expected to function as a secretion signal sequenceof 18-20 amino acids, giving a predicted size range for the members ofthis family 155 to 197 amino acids (mature MW≈17 to ≈20 kDa). Thealignment of FIG. 7 shows several conserved amino acids, including atryptophan residue and 5 cysteines in the C-terminal half of theproteins.

The PRO1031 or PRO1122 encoding nucleic acid or fragments thereof canalso be used for chromosomal localizations. For example, the chromosomelocalization of IL-17B (UNQ516) (SEQ ID NO:1) and IL-17C (UNQ561) (SEQID NO:3) was determined using Taqman primers and probes designed in the3′-untranslated regions of the IL-17B and IL-17C, was performed by PCRwith Stanford Radiation Hybrid Panel G3 panel. IL-17B (UNQ516) (SEQ IDNO:1) mapped to human chromosome 5q32-34, whereas IL-17C (UNQ561) (SEQID NO:3) was localized to chromosome 16q24. Human IL-17 itself is foundon chromosome 2q31. Rouvier et al, M. Immunol. 150: 5445 (1993).

The isolation and characterization of the two new relatives of IL-17,Applicants have established and expanded the potential role of thisfamily of cytokines may play in proinflammatory immune and otherresponses. The three members of the family, IL-17 (SEQ ID NO:1), IL-17B(SEQ ID NO:1) and IL-17C (SEQ ID NO:3), are modestly related in primarystructure with about 27% overall amino acid identity including 5conserved cysteine residues (FIG. 7). The three family members share anumber of features—they are 150-200 amino acid residues in length, theyare secreted from cells via a hydrophobic secretion signal sequence, andthey are expressed as disulfide-linked homodimers that in some casesappear to be glycosylated.

While members of the same gene family based on amino acid sequencesimilarity, the three proteins are expressed in different tissues andare dispersed in the genome. IL-17 expression (SEQ ID NO:11) has beenreported only in activated T-cells, Fossiez et al., J. Exp. Med. 183:2593 (1996), Yao et al., J. Immunol. 155: 5483 (1995)[Yao-3], while itis demonstrated herein that IL-17B (DNA59294) (SEQ ID NO:2) is expressedin normal human adult pancreas, small intestine, and stomach (FIG. 8).The expression pattern of IL-17C (DNA62377) (SEQ ID NO:4), however, ismuch more restricted, as confirmed expression in other tissues has notyet been discovered.

The characterizations described herein demonstrate that the biologicalactivity of IL-17B (UNQ516) (SEQ ID NO:1) and IL-17C (UNQ561) (SEQ IDNO:3) are considerably different from the established activities forIL-17 (SEQ ID NO:11). IL-17B (UNQ516) (SEQ ID NO:1) and IL-17C (UNQ561)(SEQ ID NO:3) each fail to induce IL-6 production in human foreskinfibroblasts (Example 10) (FIG. 9A). This is in contrast to the markedinduction known for IL-17 (SEQ ID NO:11). Yao et al., Immunity 3:811(1995) [Yao-1], Yao et al., J. Immunol. 155:5483 (1995)[Yao-3].Conversely, IL-17B (SEQ ID NO:1) and IL-17C (SEQ ID NO:3), each inducethe release of TNF-α from the monocytic cell line, THP1, while IL-17 hasonly a very small effect (FIG. 9B). The stimulated release of TNF-α inTHP1 cells by IL-17B (SEQ ID NO:1) and IL-17C (SEQ ID NO:3) is time andconcentration dependent, (Example 10) (FIG. 10), with IL-17B (SEQ IDNO:1) being about 10-fold more potent than IL-17C (SEQ ID NO:3)[EC₅₀=2.4 nM for IL-17B vs. 25 nM for IL-17C].

The different biological effects of IL-17 (SEQ ID NO:11) as compared toIL-17B or C (SEQ ID NO:s 1 &3), suggests that they may function via adifferent cell surface receptor (or some differing receptor components)than the known IL-17 receptor. Yao et al., Cytokine 9:794 (1997)[Yao-3]. In an effort to examine the question of receptor specificitydirectly, Applicants have demonstrated that both IL-17B (SEQ ID NO:1)and IL-17C (SEQ ID NO:3) fail to bind to the IL-17 receptor ECD (SEQ IDNO:16) (FIG. 11A), and also fail to compete for the binding of IL-17(SEQ ID NO:11) to its receptor ECD (SEQ ID NO:16) (FIG. 11B). IL-17B(SEQ ID NO:1) and IL-17C (SEQ ID NO:3) do bind to the surface of THP1cells, where they have activity (FIG. 12). The interaction is specificat least to the extent that a control Fc fusion protein fails to bind tothese cells. The results suggest that there could be a set of receptorsthat bind and transduce the signal from the family of IL-17 cytokines, areceptor/ligand model that has been found for many cytokine and growthfactor families.

The novel cytokines disclosed herein, PRO1031 (e.g., 516) and PRO1122(e.g., UNQ561), differ from IL-17 (SEQ ID NO:11) in their patterns ofexpression and biological activities. The differential expressioncoupled with the lack of interaction with the known IL-17 receptorsuggests and expanded role for the IL-17 family in the proinflammatoryimmune response.

F. Anti-PRO1031 and anti-PRO1122 Antibodies

The present invention further provides anti-PRO1031 and anti-PRO1122polypeptide antibodies. Exemplary antibodies include polyclonal,monoclonal, humanized, bispecific, and heteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-PRO1031 or anti-PRO1122 antibodies of the present invention maycomprise polyclonal antibodies. Methods of preparing polyclonalantibodies are known to the skilled artisan. Polyclonal antibodies canbe raised in a mammal, for example, by one or more injections of animmunizing agent and, if desired, an adjuvant. Typically, the immunizingagent and/or adjuvant will be injected in the mammal by multiplesubcutaneous or intraperitoneal injections. The immunizing agent mayinclude the PRO1031 or PRO1122 polypeptide or a fusion protein thereof.It may be useful to conjugate the immunizing agent to a protein known tobe immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-PRO1031 or anti-PRO1122 antibodies may, alternatively, bemonoclonal antibodies. Monoclonal antibodies may be prepared usinghybridoma methods, such as those described by Kohler and Milstein,Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, orother appropriate host animal, is typically immunized with an immunizingagent to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the immunizing agent.Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent will typically include the PRO1031 or PRO1122polypeptide or a fusion protein thereof. Generally, either peripheralblood lymphocytes (“PBLs”) are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell [Goding, MonoclonalAntibodies. Principles and Practice, Academic Press, (1986) pp. 59-103].Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells may becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against aPRO1031 or PRO1122 polypeptide. Preferably, the binding specificity ofmonoclonal antibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81, 6851-6855(1984)] or by covalently joining to the immunoglobulin coding sequenceall or part of the coding sequence for a non-immunoglobulin polypeptide.Such a non-immunoglobulin polypeptide can be substituted for theconstant domains of an antibody of the invention, or can be substitutedfor the variable domains of one antigen-combining site of an antibody ofthe invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

3. Humanized Antibodies

The anti-PRO1031 or anti-PRO1122 antibodies of the invention may furthercomprise humanized antibodies or human antibodies. Humanized forms ofnon-human (e.g., murine) antibodies are chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or complete inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368: 812-13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51(1996); Neuberger, Nature Biotechnology 14: 826 (1996); Lonberg andHuszar, Intern. Rev. Immunol. 13: 65-93 (1995).

4. Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

The antibodies of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g. a peptidyl chemotherapeutic agent, see WO 81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such as way so as toconvert it into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, glycosidase, glucose oxidase, human lysosyme, humanglucuronidase, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases (e.g., carboxypeptidase G2and carboxypeptidase A) and cathepsins (such as cathepsins B and L),that are useful for converting peptide-containing prodrugs into freedrugs; D-alanylcarboxypeptidases, useful for converting prodrugs thatcontain D-amino acid substituents; carbohydrate-cleaving enzymes such asβ-galactosidase and neuraminidase useful for converting glycosylatedprodrugs into free drugs; β-lactamase useful for converting drugsderivatized with β-lactams into free drugs; and penicillin amidases,such as penicillin Vamidase or penicillin G amidase, useful forconverting drugs derivatized at their amine nitrogens with phenoxyacetylor phenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as Aabzymes@can be used to convert the prodrugs of the invention into free activedrugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to theanti-PRO1031 or anti-PRO1122 antibodies by techniques well known in theart such as the use of the heterobifunctional cross-linking agentsdiscussed above. Alternatively, fusion proteins comprising at least theantigen binding region of the antibody of the invention linked to atleast a functionally active portion of an enzyme of the invention can beconstructed using recombinant DNA techniques well known in the art (see,e.g. Neuberger et al., Nature 312: 604-608 (1984)).

5. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is for aPRO1031 polypeptide, the other one is for any other antigen, andpreferably for a cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al. Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory Acavities@ of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab=)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229: 81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab=)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab=fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab=-TNB derivatives isthen reconverted to the Fab=-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab=-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab=fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecificantibody F(ab=)₂ molecule. Each Fab=fragment was separately secretedfrom E. coli and subjected to directed chemical coupling in vitro toform the bispecific antibody. The bispecific antibody thus formed wasable to bind to cells overexpressing the ErbB2 receptor and normal humanT cells, as well as trigger the lytic activity of human cytotoxiclymphocytes against human breast tumor targets.

Various technique for making and isolating bispecific antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bispecific antibodies have been produced using leucine zippers,Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992), wherein theleucine zipper peptides from the Fos and Jun proteins were linked to theFab=portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. This method can also beutilized for the production of antibody homodimers. The Adiabody@technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for makingbispecific antibody fragments. The fragments comprise a heavy-chainvariable domain (V_(H)) connected to a light-chain variable domain(V_(L)) by a linker which is too short to allow pairing between the twodomains on the same chain. Accordingly, the V_(H) and V_(L) domains ofone fragment are forced to pair with the complementary V_(L) and V_(H)domains of another fragment, thereby forming two antigen-binding sites.Another strategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See, Gruber et al.,J. Immunol. 152: 5368(1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60(1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven APro@ protein herein. Alternatively, an anti-@PRO@ protein arm maybe combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular APRO@ protein. Bispecific antibodies mayalso be used to localize cytotoxic agents to cells which express aparticular APRO@ polypeptide. These antibodies possess a APRO@-bindingarm and an arm which binds a cytotoxic agent or a radionuclide chelator,such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody ofinterest binds the APRO@ polypeptide and further binds tissue factor(TF).

6. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

7. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance the effectiveness of theantibody. For example cysteine residue(s) may be introduced in the Fcregion, thereby allowing interchain disulfide bond formation in thisregion. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al. Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989).

8. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g. an enzymatically active toxin of bacterial, fungal, plant oranimal origin, or fragments thereof, or a small molecule toxin), or aradioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active proteintoxins and fragments thereof which can be used include diphtheria Achain, nonbinding active fragments of diphtheria toxin, cholera toxin,botulinus toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin Achain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, saporin, mitogellin, restrictocin,phenomycin, enomycin and the tricothecenes. Small molecule toxinsinclude, for example, calicheamicins, maytansinoids, palytoxin andCC1065. A variety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y and¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g. avidin) which is conjugatedto a cytotoxic agent (e.g. a radionucleotide).

9. Immunoliposomes

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.81(19): 1484 (1989).

10. Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a PRO1031 or PRO1122 polypeptideidentified herein, as well as other molecules identified by thescreening assays disclosed hereinbefore, can be administered for thetreatment of various disorders in the form of pharmaceuticalcompositions.

If a PRO1031 or PRO1122 polypeptide is intracellular and wholeantibodies are used as inhibitors, internalizing antibodies arepreferred. However, lipofections or liposomes can also be used todeliver the antibody, or an antibody fragment, into cells. Whereantibody fragments are used, the smallest inhibitory fragment thatspecifically binds to the binding domain of the target protein ispreferred. For example, based upon the variable-region sequences of anantibody, peptide molecules can be designed that retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA 90: 7889-7893 (1993).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition may comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokines, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitable present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coascervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orfelatin-microcapsules and poly-(methylmethactylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidγ-ethyl-L-glutamate, non-degradable ethylene-vinylacetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)₃-hydroxylbutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanisms involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thiosulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

G. Uses for Anti-PRO1031 and Anti-PRO1122 Antibodies

The anti-PRO1031 and anti-PRO1122 antibodies of the present inventionhave various utilities. For example, anti-PRO1031 or anti-PRO1122antibodies may be used in diagnostic assays for PRO1031 or PRO1122polypeptides, e.g., detecting expression in specific cells, tissues, orserum. Various diagnostic assay techniques known in the art may be used,such as competitive binding assays, direct or indirect sandwich assaysand immunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies. A Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in the assayscan be labeled with a detectable moiety. The detectable moiety should becapable of producing, either directly or indirectly, a detectablesignal. For example, the detectable moiety may be a radioisotope, suchas ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent or chemiluminescentcompound, such as fluorescein isothiocyanate, rhodamine, or luciferin,or an enzyme, such as alkaline phosphatase, beta-galactosidase orhorseradish peroxidase. Any method known in the art for conjugating theantibody to the detectable moiety may be employed, including thosemethods described by Hunter et al., Nature, 144:945 (1962); David etal., Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth.,40:219 (1981); and Nygren, J. Histochem. and Cylochem., 30:407 (1982).

Anti-PRO1031 or anti-PRO1122 antibodies also are useful for the affinitypurification of PRO1031 or PRO1122 polypeptides, respectively, fromrecombinant cell culture or natural sources. In this process, theantibodies against a PRO1031 or PRO1122 polypeptide are immobilized on asuitable support, such a Sephadex resin or filter paper, using methodswell known in the art. The immobilized antibody then is contacted with asample containing the PRO1031 or PRO1122 polypeptide to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except the PRO1031or PRO1122 polypeptide, which is bound to the immobilized antibody.Finally, the support is washed with another suitable solvent that willrelease the PRO1031 or PRO1122 polypeptide from the antibody.

H. PRO1031, PRO1122 and IL-17 Antagonists/Agonists

This invention encompasses methods of screening compounds to identitythose that mimic the PRO1031, PRO1122 or IL017 polypeptide (agonists) orprevent the effect of the PRO1031, PRO1122 or IL-17 polypeptide(antagonists). Screening assays for antagonist drug candidates aredesigned to identity compounds that bind or complex with the PRO1031,PRO1122, IL-17 polypeptides encoded by the genes identified herein, orotherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart. For example, to screen for antagonists and/or agonists of PRO1031,PRO1122, IL-17 signaling, the assay mixture is incubated underconditions whereby, but for the presence of the candidatepharmacological agent, PRO1031, PRO1122 or IL-17 induces TNF-α releasefrom THP-1 cells with a reference activity. Alternatively, the testedactivity can be the release of nitric oxide (NO) and proteoglycans fromIL17 and/or IL-1α treated articular cartilage.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the PRO1031, PRO1122 or IL-17 polypeptide encoded by thegene identified herein or the drug candidate is immobilized on a solidphase, e.g., on a microtiter plate, by covalent or non-covalentattachments. Non-covalent attachment generally is accomplished bycoating the solid surface with a solution of the PRO1031, PRO1122 orIL-17 polypeptide and drying. Alternatively, an immobilized antibody,e.g., a monoclonal antibody, specific for the PRO1031, PRO1122 or IL-17polypeptide to be immobilized can be used to anchor it to solid surface.The assay is performed by adding the non-immobilized component, whichmay be labeled by a detectable label, to the immobilized component,which may be labeled by a detectable label, to the immobilizedcomponent, e.g., the coated surface containing the anchored component.When the reaction is complete, the non-reacted components are removed,e.g., by washing, and complexes anchored on the solid surface aredetected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

It the candidate compound interacts with but does not bind to aparticular PRO1031, PRO1132 or IL-17 polypeptide encoded by a geneidentified herein, its interaction with that polypeptide can be assayedby methods well known for detecting protein-protein interactions. Suchassays include traditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored through gradients or chromatographic columns. In addition,protein-protein interactions can be monitored by using a yeast-basedgenetic system described by Fields and co-workers (Fields and Song,Nature 340: 245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA 88:9578-9582 (1991) as disclosed by Chevray and Nathans, Proc. Natl. Acad.Sci. USA 89: 5789-5791 (1991). Many transcriptional activators, such asyeast GAL4, consist of two physically discrete modular domains, oneacting as the DNA-binding domain, while the other functions as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptide are detected withchromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a gene encoding aPRO1031, PRO1122 or IL-17 polypeptide identified herein and other intra-or extracellular components can be tested as follows: usually a reactionmixture is prepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as a positivecontrol. The binding (complex formation) between the test compound andthe intra- or extracellular component present in the mixture ismonitored as described hereinabove. The formation of a complex in thecontrol reaction(s) but not in the reaction mixture containing the testcompound indicates that the test compound interferes with theinteraction of the test compound and its reaction partner.

Antagonists may be detected by combining the PRO1031, PRO1122 or IL-17polypeptide and a potential antagonist with membrane-bound PRO1031,PRO1122 or IL-17 polypeptide receptors or recombinant receptors underappropriate conditions for a competitive inhibition assay. The PRO1031,PRO1122 or IL-17 polypeptide can be labeled, such as by radioactivity,such that the number of PRO1031, PRO1122 or IL-17 polypeptide moleculesbound to the receptor can be used to determine the effectiveness of thepotential antagonist. The gene encoding the receptor can be identifiedby numerous methods known to those of skill in the art, for example,ligand panning and FACS sorting. Coligan et al., Current Protocols inImmun. 1(2): Ch. 5 (1991). Preferably, expression cloning is employedwherein polyadenylated RNA is prepared from a cell responsive to thePRO1031 or PRO1122 polypeptide and a cDNA library created from this RNAis divided into pools and used to transfect COS cells or other cellsthat are not responsive to the PRO1031, PRO1122 or IL-17 polypeptide,respectively. Transfected cells that are grown on glass slides areexposed to labeled PRO1031, PRO1122 or IL-17 polypeptide. The PRO1031,PRO1122 or IL-17 polypeptide can be labeled by a variety of meansincluding iodination or inclusion of a recognition site for asite-specific protein kinase. Following fixation and incubation, theslides are subjected to autoradiographic analysis. Positive pools areidentified and sub-pools are prepared and re-transfected using aninteractive sub-pooling and re-screening process, eventually yielding asingle clone that encodes the putative receptor.

As an alternative approach for receptor identification, labeled PRO1031,PRO1122 or IL-17 polypeptide can be photoaffinity-linked with cellmembrane or extract preparations that express the receptor molecule.Cross-linked material is resolved by PAGE and exposed to X-ray film. Thelabeled complex containing containing the receptor can be excised,resolved into peptide fragments, and subjected to proteinmicro-sequencing. The amino acid sequence obtained from micro-sequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identity the gene encoding the putativereceptor.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeledPRO1031, PRO1122 or IL-17 polypeptide in the presence of the candidatecompound. The ability of the compound to enhance or block thisinteraction could then be removed.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin withPRO1031, PRO1122 or IL-17 polypeptide, and, in particular, antibodiesincluding, without limitation, poly- and monoclonal antibodies andantibody fragments, single-chain antibodies, anti-idiotypic antibodies,and chimeric or humanized versions of such antibodies or fragments, aswell as human antibodies and antibody fragments. Alternatively, apotential antagonist may be a closely related protein, for example, amutated form of the PRO1031, PRO1122 or IL-17 polypeptide thatrecognizes the receptor but impart no effect, thereby competitivelyinhibiting the action of the PRO1031, PRO1122 or IL-17 polypeptide.

Another potential PRO1031, PRO1122 or IL-17 polypeptide antagonist is anantisense RNA or DNA construct prepared using antisense technology,where, e.g., an antisense RNA or DNA molecule acts to block directly thetranslation of mRNA by hybridizing to targeted mRNA and preventing itstranslation into protein. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. For example, the 5′ coding portion of the polynucleotide sequence,which encodes the mature PRO1031, PRO1122 or IL-17 polypeptides herein,is used to design an antisense RNA oligonucleotide sequence, whichencodes the mature PRO1031, PRO1122 or IL-17 polypeptides herein, isused to design an antisense RNA oligonucleotide of about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription (triple helix—see Leeet al., Nucl. Acids Res. 6: 3073 (1979); Cooney et al., Science 241: 456(1988); Dervan et al., Science 251: 1360 (1991)), thereby preventingtranscription and the production of the PRO1031 or PRO1122 polypeptide.The antisense RNA oligonucleotide hybridizes to the mRNA in vivo andblocks translation of the mRNA molecule into the PRO1031, PRO1122 orIL-17 polypeptide (antisense-Okano, Neurochem. 546: 560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRCPress: Boca Raton, Fla., 1988). The oligonucleotides described above canalso be delivered to cells such that the antisense RNA or DNA may beexpressed in vivo to inhibit production of the PRO1031, PRO1122, IL-17polypeptide. When antisense DNA is used, oligodeoxyribonucleotidesderived from the translation-initiation site, e.g., between about −10and +10 positions of the target gene nucleotide sequence, are preferred.

Potential antagonists include small molecules that bind to the activesite, the receptor binding site, or growth factor or other relevantbinding site of the PRO1031 or PRO1122 polypeptide, thereby blocking thenormal biological activity of the PRO1031, PRO1122 or IL-17 polypeptide.Examples of small molecules include, but are not limited to, smallpeptides or peptide-like molecules, preferably soluble peptides, andsynthetic non-peptidyl organic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonuclytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details, see e.g., Rossi, CurrentBiology 4: 469-471 (1994) and PCT publication No. WO 97/33551 (publishedSep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCR publication No. WO 97/33551, supra.

I. Diagnostic Uses

Another use of the compounds of the invention (e.g., PRO1031- orPRO1122-variants and anti-PRO1031 or anti-PRO1122 antibodies) describedherein is to help diagnose whether a disorder is driven, to some extent,by PRO1031 or PRO1122 modulated signaling.

A diagnostic assay to determine whether a particular disorder (e.g.,degenerative cartilaginous disorder) is driven by PRO1031 or PRO1122signaling, can be carried out using the following steps: (1) culturingtest cells or tissues expressing PRO1031 or PRO1122; (2) administering acompound which can inhibit PRO1031 or PRO1122 modulated signaling; and(3) measuring the PRO1031 or PRO1122 mediated phenotypic effects in thetest cells. The steps can be carried out using standard techniques inlight of the present disclosure. For example, standard techniques can beused to isolate cells or tissues and culturing or in vivo.

Compounds of varying degree of selectivity are useful for diagnosing therole of PRO1031 or PRO1122. For example, compounds which PRO1031 orPRO1122 in addition to another form of adaptor molecule can be used asan initial test compound to determine if one of several adaptormolecules drive the disorder. The selective compounds can then be usedto further eliminate the possible role of the other adaptor proteins indriving the disorder. Test compounds should be more potent in inhibitingintracellular signaling activity than in exerting a cytotoxic effect(e.g., an IC₅₀/LD₅₀ of greater than one). The IC₅₀ and LD₅₀ can bemeasured by standard techniques, such as an MTT assay, or by measuringthe amount of LDH released. The degree of IC₅₀/LD₅₀ of a compound shouldbe taken into account in evaluating the diagnostic assay. Generally, thelarger the ratio the more relative the information. Appropriate controlstake into account the possible cytotoxic effect of a compound of acompound, such as treating cells not associated with a cellproliferative disorder (e.g., control cells) with a test compound, canalso be used as part of the diagnostic assay. The diagnostic methods ofthe invention involve the screening for agents that modulate the effectsof PRO1031 or PRO1122 upon degenerative cartilagenous disorders.Exemplary detection techniques include radioactive labeling andimmunoprecipitating (U.S. Pat. No. 5,385,915).

For example, antibodies, including antibody fragments, can be used toqualitatively or quantitatively detect the expression of proteinsencoded by the disease-related genes (Amarker gene products@). Theantibody preferably is equipped with a detectable, e.g. fluorescentlabel, and binding can be monitored by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art.

In situ detection of antibody binding to the marker gene products can beperformed, for example, by immunofluorescence or immunoelectronmicroscopy. For this purpose, a histological specimen is removed fromthe patient, and a labeled antibody is applied to it, preferably byoverlaying the antibody on a biological sample. This procedure alsoallows for determining the distribution of the marker gene product inthe tissue examined. It will be apparent for those skilled in the artthat a wide variety of histological methods are readily available for insitu detection.

J. Pharmaceutical Compositions

The PRO1031 or PRO1122, antagonists or agonists thereof (e.g.,antibodies), as well as other molecules identified by the screeningassays disclosed hereinbefore, can be employed as therapeutic agents.Such therapeutic agents are formulated according to known methods toprepare pharmaceutically useful compositions, whereby the PRO1031 orPRO1122, antagonist or agonist thereof is combined in admixture with apharmaceutically acceptable carrier.

In the case of PRO1031 or PRO1122 antagonist or agonist antibodies, ifthe protein encoded by the amplified gene is intracellular and wholeantibodies are used as inhibitors, internalizing antibodies arepreferred. However, lipofections or liposomes can also be used todeliver the antibody, or an antibody fragment, into cells. Whereantibody fragments are used, the smallest inhibitory fragment whichspecifically binds to the binding domain of the target protein ispreferred. For example, based upon the variable region sequences of anantibody, peptide molecules can be designed which retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology (see, e.g.Marasco et al., Proc. Natl. Acad. Sci. USA 90: 7889-7893 [1993]).

Therapeutic formulations are prepared for storage by mixing the activeingredient having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition may comprise a cytotoxicagent, cytokine or growth inhibitory agent. Such molecules are suitablypresent in combination in amounts that are effective for the purposeintended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, and intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andγ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.Microencapsulation of recombinant proteins for sustained release hasbeen successfully performed with human growth hormone (rhGH),interferon-(rhlFN-), interleukin-2, and MN rpg 120. Johnson et al., Nat.Med. 2: 795-799 (1996); Yasuda et al., Biomed. Ther. 27: 1221-1223(1993); Hora et al., Bio/Technology 8: 755-758 (1990); Cleland, “Designand Production of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds., (Penum Press: New York,1995), pp. 439-462; WO 97/03692; WO 96/40072; WO 96/07399; and U.S. Pat.No. 5,654,010.

The sustained-release formulations of these proteins may be developedusing poly lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer”, in Biodegradable Polymers as DrugDelivery Systems (Marcel Dekker; New York, 1990), M. Chasin and R.Langer (Eds.) pp. 1-41.

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

K. Methods of Treatment

It is contemplated that the compounds of the present invention may beused to treat various conditions, including those characterized byoverexpression and/or activation of the disease-associated genesidentified herein. Exemplary conditions or disorders to be treated withsuch antibodies and other compounds, including, but not limited to,small organic and inorganic molecules, peptides, antisense molecules,etc. include inflammatory and immunologic disorders, especially thosecharacterized by cartilage matrix breakdown such as arthritis, (e.g.,osteoarthritis, psoriatic arthritis, rheumatoid arthritis) or otherdegenerative inflammatory diseases.

The active agents of the present invention, e.g. antibodies, areadministered to a mammal, preferably a human, in accord with knownmethods, such as intravenous administration as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerebral, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, intraoccular, intralesional, oral, topical,inhalation or through sustained release.

Other therapeutic regimens may be combined with the administration ofthe PRO1031, PRO1122, antagonists or antagonists, anti-cancer agents,e.g. antibodies of the instant invention.

For the prevention or treatment of disease, the appropriate dosage of anactive agent, (e.g. an antibody) will depend on the type of disease tobe treated, as defined above, the severity and course of the disease,whether the agent is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the agent, and the discretion of the attending physician. The agentis suitably administered to the patient at one time or over a series oftreatments.

Dosages and desired drug concentration of pharmaceutical compositions ofthe present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary artisan. Animalexperiments provide reliable guidance for the determination of effectivedoes for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” InToxicokinetics and New Drug Development, Yacobi et al., Eds, PergamonPress, New York 1989, pp. 42-46.

When in vivo administration of a PRO1031 or PRO1122 polypeptide oragonist or antagonist thereof is employed, normal dosage amounts mayvary from about 10 μg/kg up to 100 mg/kg of mammal body weight or moreper day, preferably about 1 μg/kg/day up to 100 mg/kg of mammal bodyweight or more pre day, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature; see, for example, U.S. Pat. Nos. 4,657,760, 5,206,344 or5,255,212. It is within the scope of the invention that differentformulations will be effective for different treatment compounds anddifferent disorders, that administration targeting one organ or tissue,for example, may necessitate delivery in a manner different from that toanother organ or tissue. Moreover, dosages may be administered by one ormore separate administrations, or by continuous infusion. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

L. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the diagnosis or treatment of thedisorders described above is provided. The article of manufacturecomprises a container and a label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is effective for diagnosing ortreating the condition and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). The active agentin the composition is typically a PRO1031, PRO1122 polypeptide,antagonist, or agonist thereof. The label on, or associated with, thecontainer indicates that the composition is used for diagnosing ortreating the condition of choice. The article of manufacture may furthercomprise a second container comprising a pharmaceutically-acceptablebuffer, such as phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Rockville, Md.

Example 1 Isolation of cDNA Clones Encoding Human PRO1031

The extracellular domain (ECD) sequences (including the secretionsignal, if any) of from about 950 known secreted proteins from theSwiss-Prot public protein database were used to search expressedsequence tag (EST) databases. The EST databases included public ESTdatabases (e.g., GenBank, Merck/Wash U.) and a proprietary EST DNAdatabase (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.). Thesearch was performed using the computer program BLAST or BLAST2 (Altshulet al., Methods in Enzymology 266:460-480 (1996)) as a comparison of theECD protein sequences to a 6 frame translation of the EST sequence.Those comparisons resulting in a BLAST score of 70 (or in some cases 90)or greater that did not encode known proteins were clustered andassembled into consensus DNA sequences with the program “phrap” (PhilGreen, University of Washington, Seattle, Wash.).

An initial virtual sequence fragment (consensus assembly) was assembledrelative to other EST sequences using phrap. The initial consensus DNAsequence was extended using repeated cycles of BLAST and phrap to extendthe consensus sequence as far as possible using the sources of ESTsequences discussed above. The results of this assembly is shown in FIG.5 (SEQ ID NO:5), also referred to as DNA47332.

One sequence comprising the consensus assembly, W74558 (clone 344649)(SEQ ID NO:6) was further examined. The sequence was obtained from theIMAGE consortium and analyzed. Lennon et al., Genomics 33: 151 (1996).DNA sequencing gave the full-length DNA sequence for PRO1031 [hereindesignated as DNA59294-1381] (SEQ ID NO:2) and the derived PRO1031protein sequence (UNQ516) (SEQ ID NO:1).

The entire nucleotide sequence of DNA59294-1381 is shown in FIG. 2 (SEQID NO:2). Clone DNA59294-1381 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 42-44and ending at the stop codon at nucleotide positions 582-584 (FIG. 2)(SEQ ID NO:2). The predicted polypeptide precursor is 180 amino acidslong (FIG. 1) (SEQ ID NO:1). The full-length PRO1031 (UNQ516) proteinshown in FIG. 2 (SEQ ID NO:1) has an estimated molecular weight of about20437 and a pI of about 9.58. Clone DNA59294-1381 (SEQ ID NO:2) has beendeposited with the ATCC, and have been assigned deposit number 209866.In the event of any sequencing irregularities or errors with thesequences provided herein, it is understood that the deposited clonecontains the correct sequence for DNA59624 (SEQ ID NO:2). Furthermore,the sequences provided herein are the result of known sequencingtechniques.

Analysis of the amino acid sequence of the full-length PRO1031polypeptide (UNQ516) (SEQ ID NO:1) suggests that it is a novel cytokine.

Further analysis of the amino acid sequence of SEQ ID NO:2 reveals thatthe putative signal peptide is at about amino acids 1-20 of SEQ ID NO:2.An N-glycosylation site is at about amino acids 75-78 of SEQ ID NO:2. Aregion having sequence identity with IL-17 is at about amino acids96-180. The corresponding nucleotides can be routinely determined giventhe sequences provided herein.

Example 2 Isolation of cDNA Clones Encoding Human PRO1122

An expressed sequence tag (EST) DNA database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.) was searched and an EST wasidentified. The EST was Incyte 1347523 (SEQ ID NO:7) also calledDNA49665. Based on DNA49665 (SEQ ID NO:7), oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolated a clone ofthe full-length coding sequence for the PRO1122. [e.g., Sambrook et al.,Molecular Cloning. A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989); Dieffenbach et al., PCR Primer: A LaboratoryManual (Cold Spring Harbor Laboratory Press, 1995)].

Forward and reverse PCR primers generally range from 20 to 30nucleotides and are often designed to give a PCR product of about100-1000 bp in length. The probes sequences are typically 40-55 bp inlength. In some cases, additional oligonucleotides are synthesized whenthe consensus sequence is greater than about 1-1.5 kpb. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausuble et al., CurrentProtocols in Molecular Biology, with the PCR primer pair. A positivelibrary was then used to isolate clones encoding the gene of interestusing the probe oligonucleotide and one of the primer pairs.

PCR primers (forward, reverse and hybridization) were synthesized:forward PCR primer: 5′-ATCCACAGAAGCTGGCCTTCGCCG-3′ (SEQ ID NO:8) reversePCR primer: 5′-GGGACGTGGATGAACTCGGTGTGG-3′ (SEQ ID NO:9)

hybridization probe: (SEQ ID NO:10)5′-TATCCACAGAAGCTGGCCTTCGCCGAGTGCCTGTGCAGAG-3′.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO1122 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue. The cDNA libraries used to isolate the cDNA clones wereconstructed using standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science 235:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO1122 [herein designated asDNA62377-1381-1] (SEQ ID NO:4) and the derived protein PRO1122 sequence(UNQ561) (SEQ ID NO:3).

The entire nucleotide sequence of DNA62377-1381-1 (SEQ ID NO:4) is shownin FIGS. 4A-4B (SEQ ID NO:4). Clone DNA62377-1381-1 (SEQ ID NO:4)contains a single open reading frame with an apparent translationalinitiation site at nucleotide positions 50-52 and ending at the stopcodon at nucleotide positions 641-643 of SEQ ID NO:4 (FIGS. 4A-4B). Thepredicted polypeptide precursor is 197 amino acids long (FIG. 3) (SEQ IDNO:3). The full-length PRO1122 protein shown in FIG. 3 (UNQ561) (SEQ IDNO:3) has an estimated molecular weight of about 21765 daltons and a pIof about 8.53. Clone DNA62377-1381-1 has been deposited with the ATCC onDec. 22, 1998 and has been assigned deposit number 203552. It isunderstood that in the event or a sequencing irregularity or error inthe sequences provided herein, the correct sequence is the sequencedeposited. Futhermore, all sequences provided herein are the result ofknown sequencing techniques.

Analysis of the amino acid sequence of the isolated full-length PRO1122(UNQ561) suggests that it possesses similarity with IL-17, therebyindicating that PRO1122 (UNQ561) may be a novel cytokine. FIG. 3 (SEQ IDNO:3) also shows the approximate locations of the signal peptide,leucine zipper pattern, and a region having sequence identity withIL-17. The corresponding nucleotides can be routinely determined, e.g.,by reference to FIGS. 4A-4B.

Example 3 Use of PRO1031- or PRO1122-Encoding DNA as a HybridizationProbe

The following method describes use of a nucleotide sequence encodingPRO1031 as a hybridization probe.

DNA comprising the coding sequence of full-length PRO1031 (as shown inFIG. 2, SEQ ID NO:2), PRO1122 (as shown in FIG. 4, SEQ ID NO:4) or afragment thereof is employed as a probe to screen for homologous DNAs(such as those encoding naturally-occurring variants of PRO1031 orPRO1122 in human tissue cDNA libraries or human tissue genomiclibraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled PRO1031 or PRO1122 polypeptide-derived probe to thefilters is performed in a solution of 50% formamide, 5×SSC, 0.1% SDS,0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2× Denhardt'ssolution, and 10% dextran sulfate at 42° C. for 20 hours. Washing of thefilters is performed in an aqueous solution of 0.1×SSC and 0.1% SDS at42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO1031 or PRO1122 polypeptide can then beidentified using standard techniques known in the art.

Example 4 Expression of PRO1031 or PRO1122 Polypeptides in E. coli

This example illustrates the preparation of unglycosylated forms ofPRO1031 or PRO1122 polypeptides by recombinant expression in E. coli.

The DNA sequence encoding the full-length PRO1031, PRO1122 or a fragmentor variant thereof is initially amplified using selected PCR primers.The primers should contain restriction enzyme sites which correspond tothe restriction enzyme sites on the selected expression vector. Avariety of expression vectors may be employed. An example of a suitablevector is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95(1977)) which contains genes for ampicillin and tetracycline resistance.The vector is digested with restriction enzyme and dephosphorylated. ThePCR amplified sequences are then ligated into the vector. The vectorwill preferably include sequences which encode for an antibioticresistance gene, a trp promoter, a polyhis leader (including the firstsix STII codons, polyhis sequence, and enterokinase cleavage site), thePRO1031 or PRO1122 coding region, lambda transcriptional terminator, andan argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO1031 or PRO1122 polypeptide can then be purifiedusing a metal chelating column under conditions that allow tight bindingof the polypeptide.

Example 5 Expression of PRO1031 or PRO1122 Polypeptides in MammalianCells

This example illustrates preparation of glycosylated forms of PRO1031 orPRO1122 polypeptides by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the PRO1031- or PRO1122-encodingDNA is ligated into pRK5 with selected restriction enzymes to allowinsertion of the PRO1031- or PRO1122-encoding DNA using ligation methodssuch as described in Sambrook et al., supra. The resulting vector iscalled pRK5-PRO1031 or pRK5-PRO1122, respectively.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μgpRK5-PRO1031 or pRK5-PRO1122 DNA is mixed with about 1 μg DNA encodingthe VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolvedin 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixtureis added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mMNaPO₄, and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of PRO1031 or PRO1122 polypeptide. The cultures containingtransfected cells may undergo further incubation (in serum free medium)and the medium is tested in selected bioassays.

In an alternative technique, PRO1031- or PRO1122-encoding DNA may beintroduced into 293 cells transiently using the dextran sulfate methoddescribed by Somparyrac et al., Proc. Nail. Acad. Sci., 12:7575 (1981).293 cells are grown to maximal density in a spinner flask and 700 μgpRK5-PRO1031 or pRK5-PRO1122 DNA is added. The cells are firstconcentrated from the spinner flask by centrifugation and washed withPBS. The DNA-dextran precipitate is incubated on the cell pellet forfour hours. The cells are treated with 20% glycerol for 90 seconds,washed with tissue culture medium, and re-introduced into the spinnerflask containing tissue culture medium, 5 μg/ml bovine insulin and 0.1μg/ml bovine transferrin. After about four days, the conditioned mediais centrifuged and filtered to remove cells and debris. The samplecontaining expressed PRO1031 or PRO1122 polypeptide can then beconcentrated and purified by any selected method, such as dialysisand/or column chromatography.

In another embodiment, PRO1031 or PRO1122 polypeptide can be expressedin CHO cells. The pRK5-PRO1031 or pRK5-PRO1122 vector can be transfectedinto CHO cells using known reagents such as CaPO₄ or DEAE-dextran. Asdescribed above, the cell cultures can be incubated, and the mediumreplaced with culture medium (alone) or medium containing a radiolabelsuch as ³⁵S-methionine. After determining the presence of PRO1031 orPRO1122 polypeptide, the culture medium may be replaced with serum freemedium. Preferably, the cultures are incubated for about 6 days, andthen the conditioned medium is harvested. The medium containing theexpressed PRO1031 or PRO1122 polypeptide can then be concentrated andpurified by any selected method.

Epitope-tagged PRO1031 or PRO1122 polypeptide may also be expressed inhost CHO cells. The PRO1031- or PRO1122-encoding DNA may be subclonedout of the pRK5 vector. The subclone insert can undergo PCR to fuse inframe with a selected epitope tag such as a poly-his tag into aBaculovirus expression vector. The poly-his tagged PRO1031- orPRO1122-encoding DNA insert can then be subcloned into a SV40 drivenvector containing a selection marker such as DHFR for selection ofstable clones. Finally, the CHO cells can be transfected (as describedabove) with the SV40 driven vector. Labeling may be performed, asdescribed above, to verify expression. The culture medium containing theexpressed poly-His tagged PRO1031 or PRO1122 polypeptide can then beconcentrated and purified by any selected method, such as byNi²⁺-chelate affinity chromatography.

Example 6 Expression of a PRO1031 Polypeptide in Yeast

The following method describes recombinant expression of PRO1031 orPRO1122 polypeptides in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO1031 or PRO1122 polypeptide from theADH2/GAPDH promoter. DNA encoding the PRO1031 or PRO1122 polypeptide ofinterest, a selected signal peptide and the promoter is inserted intosuitable restriction enzyme sites in the selected plasmid to directintracellular expression of the PRO1031 or PRO1122 polypeptide. Forsecretion, DNA encoding the PRO1031 or PRO1122 polypeptide can be clonedinto the selected plasmid, together with DNA encoding the ADH2/GAPDHpromoter, the yeast alpha-factor secretory signal/leader sequence, andlinker sequences (if needed) for expression of the PRO1031 or PRO1122polypeptide.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant PRO1031 or PRO1122 polypeptide can subsequently be isolatedand purified by removing the yeast cells from the fermentation medium bycentrifugation and then concentrating the medium using selectedcartridge filters. The concentrate containing the PRO1031 or PRO1122polypeptide may further be purified using selected column chromatographyresins.

Example 6 Expression of PRO1031 or PRO1122 Polypeptides inBaculovirus-Infected Insect Cells

The following method describes recombinant expression of PRO1031 orPRO1122 polypeptides in Baculovirus-infected insect cells.

The PRO1031- or PRO1122-encoding DNA is fused upstream of an epitope tagcontained within a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thePRO1031- or PRO1122-encoding DNA or the desired portion of the PRO1031-or PRO1122-encoding DNA (such as the sequence encoding the extracellulardomain of a transmembrane protein) is amplified by PCR with primerscomplementary to the 5′ and 3′ regions. The 5′ primer may incorporateflanking (selected) restriction enzyme sites. The product is thendigested with those selected restriction enzymes and subcloned into theexpression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4 to 5 days of incubation at 28° C.,the released viruses are harvested and used for further amplifications.Viral infection and protein expression is performed as described byO'Reilley et al., Baculovirus Expression vectors: A Laboratory Manual,Oxford:Oxford University Press (1994).

Expressed poly-his tagged PRO1031 or PRO1122 polypeptide can then bepurified, for example, by Ni²⁺-chelate affinity chromatography asfollows. Extracts are prepared from recombinant virus-infected Sf9 cellsas described by Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9;12.5 mM MgCl₂; 0.1 mM EDTA; 10% Glycerol; 0.1% NP-40; 0.4 M KCl), andsonicated twice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7.8) and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% Glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged PRO1031 polypeptide are pooled anddialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) PRO1031polypeptide can be performed using known chromatography techniques,including for instance, Protein A or protein G column chromatography.

Example 7 Preparation of Antibodies that Bind PRO1031 or PRO1122Polypeptides

This example illustrates the preparation of monoclonal antibodies whichcan specifically bind to PRO1031 or PRO1122 polypeptides.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified PRO1031 or PRO1122 polypeptide, fusionproteins containing a PRO1031 or PRO1122 polypeptide, and cellsexpressing recombinant PRO1031 or PRO1122 polypeptide on the cellsurface. Selection of the immunogen can be made by the skilled artisanwithout undue experimentation.

Mice, such as Balb/c, are immunized with the PRO1031 or PRO1122immunogen emulsified in complete Freund's adjuvant and injectedsubcutaneously or intraperitoneally in an amount from 1-100 micrograms.Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (RibiImmunochemical Research, Hamilton, Mont.) and injected into the animal'shind foot pads. The immunized mice are then boosted 10 to 12 days laterwith additional immunogen emulsified in the selected adjuvant.Thereafter, for several weeks, the mice may also be boosted withadditional immunization injections. Serum samples may be periodicallyobtained from the mice by retro-orbital bleeding for testing in ELISAassays to detect anti-PRO1031 or anti-PRO1122 polypeptide antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO1031 or PRO1122 polypeptide. Three to four days later,the mice are sacrificed and the spleen cells are harvested. The spleencells are then fused (using 35% polyethylene glycol) to a selectedmurine myeloma cell line such as P3X63AgU.1, available from ATCC, No.CRL 1597. The fusions generate hybridoma cells which can then be platedin 96 well tissue culture plates containing HAT (hypoxanthine,aminopterin, and thymidine) medium to inhibit proliferation of non-fusedcells, myeloma hybrids, and spleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstPRO1031 or PRO1122 polypeptide. Determination of “positive” hybridomacells secreting the desired monoclonal antibodies against a PRO1031 orPRO1122 polypeptide is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-PRO1031 oranti-PRO1122 polypeptide monoclonal antibodies. Alternatively, thehybridoma cells can be grown in tissue culture flasks or roller bottles.Purification of the monoclonal antibodies produced in the ascites can beaccomplished using ammonium sulfate precipitation, followed by gelexclusion chromatography. Alternatively, affinity chromatography basedupon binding of antibody to protein A or protein G can be employed.

Example 8 RNA Expression

Multi-tissue blots containing poly A⁺ RNA (2 μg per lane) from varioushuman tissues were purchased from Clontech (Palo Alto, Calif.). Theentire coding regions of human IL-17B (UNQ516) (700 bp) (SEQ ID NO:17)and IL-17C (UNQ561) (1.1 kbp) (SEQ ID NO:18) were used as hybridizationprobes. DNA probes were labeled with [α-³²P]-dCTP by random priming DNAlabeling beads (Pharmacia Biotech). Hybridization was performed usingExpresshyb (Clontech) containing the radiolabeled probes at 68° C. for 1hour. The blots were then washed with 2×SSC/0.05% SDS solution at roomtemperature for 40 minutes, followed by washes in 0.1×SSC/0.05% SDSsolution at 55° C. for 40 minutes with one change of fresh solution. Theblots were exposed in a phosphorimager and the resulting image isreported herein as FIG. 8.

For IL-17B (SEQ ID NO:1), an 800 bp mRNA transcript was found inpancreas, small intestine, and stomach of adult human tissues; a weakerband was detected in testis. (FIG. 8). IL-17C (SEQ ID NO:2) expressionwas examined in the same set of adult human tissues, but no detectablesignals were observed.

Example 9 Generating Fc/His Fusion Proteins

The coding sequences of IL17B (SEQ ID NO:17) and IL17C (SEQ ID NO:18)were amplified by PCR and subcloned into the EcoRI and SmaI sites ofpBPH.His.c to generate a C-terminal GHHHHHHHH tag (SEQ ID NO:19) or theEcoRI and Stu sites of pBPH.IgG to generate a C-terminal fusion with theFc region of human IgG1. Vectors pBPH.His.c and pBPH.IgG are derivativesof the baculovirus expression vector pVL1393 (Pharmingen). A control Fcor his-tagged protein was constructed in a similar way be C-terminallylinking pancreatitis-associated protein (175 amino acid) to the Fcportion of the human IgG1 or a his8 tag.

The fusion proteins were expressed in H5 cells using the manufacturer'srecommended procedure (Invitrogen). In brief, the DNA constructs wereco-transfected with BaculoGold Baculovirus DNA (Pharmingen) in a 7:1ratio into adherent Sf9 cells. Cells were incubated at 28° C. for 4 daysand the supernatent was harvested. The transfection supernatant wasamplified and was subject to affinity purification by either proteinA-sepharose beads (Pharmacia) for Fc fusion proteins or Ni—NTA agarosebeads (QIAGEN) for His-tagged proteins.

To examine the protein expression, SDS-PAGE analysis was performed onthe affinity purified recombinant proteins under non-reducing andreducing conditions, followed by silver staining.

Example 10 Induction of IL-6 and TNF-α Release

Using the procedure outlined in Yao et al., J. Immunol. 155: 5483 (1995)(Yao-2) for IL-6 (SEQ ID NO:14) release, human foreskin fibroblast cells(ATCC CRL-2091) were cultured in MEM media (10% FBS) with the testcytokine. After incubation for 18 hours at 37° C. and 5% CO₂,conditioned media were assayed for IL-6 using an ELISA kit (R&DSystems). For TNF-α secretion, human leukemia monocytic THP-1 cells werecultured in RPMI media (10% FBS) with test cytokine. After incubationfor 18 hour at 37° C. and 5% CO₂, conditioned media were quantitated forTNF-α (SEQ ID NO:20) using and ELISA assay kit (R&D Systems).

Human foreskin fibroblast cells (ATCC) were separately cultured in MEMmedia (10% FBS) in the presence of IL-17B (UNQ516) (SEQ ID NO:1) andIL-17C (UNQ561) (SEQ ID NO:3). After incubation for 18 hours at 37° C.and 5% CO₂, conditioned media were assayed for IL-6 (SEQ ID NO:14) usingan ELISA kit (R&D Systems). In contrast to the high level of IL-6 (SEQID NO:14) induced by IL-17 (SEQ ID NO:11), both IL-17B (SEQ ID NO:1) andIL17C (SEQ ID NO:3) failed to stimulate IL-6 (SEQ ID NO:14) secretion infibroblast cells (FIG. 9A).

Using the procedure outlined in Yao et al, Cytokine 9: 794 (1997)[Yao-3], a human leukemic monocytic cell line, THP-1, was used to assayfor the stimulation of TNF-α (SEQ ID NO:20) release by IL-17 (SEQ IDNO:11), UNQ516 (SEQ ID NO:1) and UNQ561 (SEQ ID NO:3) by culturing inRPMI media (10% FBS). After incubation for 18 hour at 37° C. and 5% CO₂,conditioned media were quantitated for TNF-α (SEQ ID NO:19) using andELISA assay kit (R&D Systems). While IL-17 (SEQ ID NO:11) induced only alow level of TNF-α (SEQ ID NO:19) in THP-1 cells, both IL-17B and IL-17C(as Fc fusion proteins) stimulated TNF-α production in THP-1 cells (FIG.9B). A control Fc fusion protein had no effect.

In order to further characterize the stimulation of TNF-α release byIl-17B and IL-17C, the time course and concentration dependence of theresponse were assayed in THP-1 cells. FIG. 10 illustrates that IL-17B(UNQ516) (SEQ ID NO:1) and IL-17C (UNQ561) (SEQ ID NO:3) stimulate therelease of TNF-α (SEQ ID NO:19) in a time- and concentration-dependentmanner. The EC₅₀ for IL-17B (UNQ516) (SEQ ID NO:1) stimulation is 2.4nM, while for IL-17C (UNQ561) (SEQ ID NO:3), 25 nM.

While the IL-17B (UNQ516) (SEQ ID NO:1) and IL-17C (UNQ561) (SEQ IDNO:3) preparations used in these experiments contained undetectablelevel of endotoxin (less than 1 EU/ml), additional control experimentswere performed to confirm that the TNF-α (SEQ ID NO:19) release fromTHP-1 cells was real and not artifactual. The IL-17B (UNQ516) (SEQ IDNO:1) and IL-17C (UNQ561) (SEQ ID NO:3) activities were unaffected bypolymyxin B treatment and were abolished by heat treatment, furthersupporting the notion that the proteins themselves were responsible forthe activities and not any contaminating endotoxin.

Example 11 IL-17 Receptor Binding

Cloning of the ECD of hIL-17 Receptor:

In order to clone the ECD of the human IL-17 receptor, twooligonucleotide primers were designed at the 5′ and 3′ ends of IL-17RECD (SEQ ID NO:15) based on the published sequence. Yao et al., supra(Yao-3). The two probes had the following sequences: primer 1: 5′-CTGTAC CTC GAG GGT GCA GAG-3′ (SEQ ID NO:20) primer 2: 5′-CCC AAG CTT GGGTCA ATG ATG ATG (SEQ ID NO:21) ATG ATG ATG ATG ATG CCA CAG GGG CAT GTAGTC C-3′

The above primers were used in PCR reactions to amplify the full-lengthcDNA from a human testis cDNA library with Pfu Turbo DNA polymerase(Promega). A C-terminal his tag was introduced by PCR through theaddition of nucleotides encoding eight histidines to the 3′ end primer.The PCR product was then subcloned into an expression plasmid vectorpRK5B. Sequence analysis confirmed that the insert contains a DNAfragment encoding the extracellular domain (1-320 amino acids) of thepublished hIL-17 receptor. (SEQ ID NO:15).

Immunoprecipitation of the IL-17R ECD:

The differential activity of IL-17 when compared to IL-17B (UNQ516) (SEQID NO:1) and IL-17C (UNQ561) (SEQ ID NO:3) suggested that they mightbind and activate different cell surface receptors. In order to testwhether IL-17B (UNQ516) (SEQ ID NO:1) or IL-17C (UNQ561) (SEQ ID NO:3)directly bind to the receptor, an expression plasmid containing theIL-17R(C-terminal his-tagged) (SEQ ID NO:22) was transfected into 293cells using SuperFect transfection reagent (Quiagen). Metabolic labelingof 293 cells was performed 16 hours after transfection using 50 μCi/ml[³⁵S]-Cys/Met mixture for 6 hours. Conditioned medium was collected andconcentrated (Centricon-10, Amicon). To examine the expression of theIL-17R ECD (SEQ ID NO:15), Ni—NTA beads (Quiagen) were used to affinityprecipitate the his-tagged IL-17R ECD (SEQ ID NO:22) from theconditioned medium.

The conditioned medium was diluted in RIPA buffer (1% NP40, 0.5% sodiumdeoxycholate, 0.1% SDS in PBS) was incubated with IL-17 (SEQ ID NO:11)and the Fc fusion proteins overnight at 4° C. Protein A-agarose beads(Pierce) were added to precipitate the Fc fusion proteins. Theprecipitates were washed three times to precipitate the Fc fusionproteins. The precipitates were washed three times in RIPA buffer,denatured in SDS sample buffer, and electrophoresed on NuPAGE 4-12%Bis-Tris gels (Novex). For IL-17 (SEQ ID NO: 11) immunoprecipitation,anti-IL-17 antibody (R&D Systems) was added. In a competitive bindingexperiment, immunoprecipitation of IL-17R ECD (SEQ ID NO:15) by IL-17(SEQ ID NO:11) is performed in the presence of a 5-fold molar excess ofIL-17B.his (SEQ ID NO:23, IL-17C.his (SEQ ID NO:24 and control histagged protein.

The IL-17R ECD (SEQ ID NO:15 migrated as a 60 kDa band when purified viaits histidine tag (FIG. 11A, lane 1). Furthermore, the IL-17R ECD (SEQID NO:15 also precipitated in combination with IL-17 (SEQ ID NO:11)(lane 3). However, both IL-17B (SEQ ID NO:1) and IL-17C (SEQ ID NO:3)failed to compete for the binding of IL-17 (SEQ ID NO:11) for thelabeled IL-17 receptor ECD (SEQ ID NO:15 (FIG. 11B, lane 15 and 16).

Example 12 Fluorescence-Activated Cell Sorter (FACS) Analysis of Bindingto THP-1 Cells

THP-1 cells (5×105) were pre-incubated in PBS containing 5% horse serumat 4° C. for 30 minutes to block non-specific binding. IL-17 (SEQ IDNO:11), IL-17B.fc (SEQ ID NO:12), IL-17C.Fc (SEQ ID NO:13), or controlFc (1 μg each) were added and incubated with the THP-1 cells in a volumeof 0.25 ml on ice for 1 hour. For the IL-17 binding experiment, primaryanti hIL-17 antibody (1:100 dilution) and secondary goat anti-mouseantibody conjugated to FITC (Jackson Immunology Lab, 1:100 dilution)were added sequentially with 30-60 minutes incubation and extensivewashes before each addition. For the Fc fusion proteins, the cells werestained with FITC conjugated goat anti-human IgG (Fc specific, JacksonImmunology Lab, 1:100 dilution). After thorough washes, a minimum of5,000 cells were analyzed using a FACScan (Becton Dickinson).

The resulting of the above procedure was that both IL-17B (SEQ ID NO:12)and IL-17C (SEQ ID NO:13) Fc fusion proteins displayed binding to THP-1cells compared with a control Fc fusion protein (FIG. 13).

Example 13 Purification of PRO1031 or PRO1122 Polypeptides UsingSpecific Antibodies

Native or recombinant PRO1031 or PRO1122 polypeptides may be purified bya variety of standard techniques in the art of protein purification. Forexample, pro-PRO1031 or pro-PRO1122 polypeptide, mature PRO1031 orPRO1122 polypeptide, or pre-PRO1031 or pre-PRO1122 polypeptide ispurified by immunoaffinity chromatography using antibodies specific forthe PRO1031 or PRO1122 polypeptide of interest. In general, animmunoaffinity column is constructed by covalently coupling theanti-PRO1031 or anti-PRO1122 antibody to an activated chromatographicresin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE® (Pharacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column is utilized in the purification of PRO1031or PRO1122 polypeptide by preparing a fraction of cells containingPRO1031 or PRO1122 polypeptide in a soluble form. This preparation isderived by solubilization of the whole cell or of a subcellular fractionobtained via differential centrifugation by the addition of detergent orby other methods well known in the art. Alternatively, soluble PRO1031or PRO1122 polypeptide containing a signal sequence may be secreted inuseful quantity into the medium in which the cells are grown.

A soluble PRO1031 or PRO1122 polypeptide-containing preparation ispassed over the immunoaffinity column, the and column is washed underconditions that allow the preferential absorbance of PRO1031 or PRO1122polypeptide (e.g., high ionic strength buffers in the presence ofdetergent). Then, the column is eluted under conditions that disruptantibody-PRO1031 or -PRO1122 polypeptide binding (e.g., a low pH buffersuch as approximately pH 2-3, or a high concentration of a chaotropesuch as urea or thiocyanate ion), and PRO1031 or PRO1122 polypeptide iscollected.

Example 14 Drug Screening

This invention is particularly useful for screening compounds by usingPRO1031 or PRO1122 polypeptides or binding fragments thereof in any of avariety of drug screening techniques. The PRO1031 or PRO1122 polypeptideor fragment employed in such a test may either bye free in solution,affixed to a solid support, borne on a cell surface, or locatedintracellularly. One method of drug screening utilized eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the PRO1031 or PRO1122 polypeptide or fragment.Drugs are screened against such transformed cells in competitive bindingassays. Such cells, either in viable or fixed form, can be used forstandard binding assays. On may measure, for example, the formation ofcomplexes between PRO1031 or PRO1122 polypeptide or a fragment and theagent being tested. Alternatively, one can examine the diminution incomplex formation between the PRO1031 or PRO1122 polypeptide and itstarget cell or target receptors caused by the agent being tested.

Thus, the present invention provides methods of screening for drugs orany other agents which can affect a PRO1031 or PRO1122polypeptide-associated disease or disorder. These methods comprisecontacting such an agent with a PRO1031 or PRO1122 polypeptide orfragment thereof and assaying (i) for the presence of a complex betweenthe agent and the PRO1031 or PRO1122 polypeptide or fragment, or (ii)for the presence of a complex between the PRO1031 or PRO1122 polypeptideor fragment and the cell, by methods well known in the art. In suchcompetitive binding assays, the PRO1031 or PRO1122 polypeptide orfragment is typically labeled. After suitable incubation, free PRO1031or PRO1122 polypeptide or fragment is separated from that present inbound form, and the amount of free or uncomplexed label is a measure ofthe ability of the particular agent to bind to PRO1031 or PRO1122 or tointerfere with the PRO1031 or PRO1122 polypeptide/cell complex.

Another technique for drug screening provide high throughput screeningfor compounds having suitable binding affinity to a polypeptide and isdescribed in detail in WO 84/03564, published on Sep. 13, 1984. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. As applied to a PRO1031 or PRO1122 polypeptide, the peptidetest compounds are reacted with PRO1031 or PRO1122 polypeptide andwashed. Bound PRO1031 or PRO1122 is detected by methods well known inthe art. Purified PRO1031 or PRO1122 polypeptide can also be coateddirectly onto plates for use in the aforementioned drug screeningtechniques. In addition, non-neutralizing antibodies can by used tocapture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding PRO1031 orPRO1122 polypeptide specifically compete with a test compound forbinding to PRO1031 or PRO1122 polypeptide or fragments thereof. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with PRO1031 or PRO1122polypeptide.

Example 15 Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptide of interest (i.e., a PRO1031 or PRO1122polypeptide) or of small molecules with which they interact, e.g.,agonists, antagonists, or inhibitors. Any of these examples can be usedto fashion drugs which are more active or stable forms of the PRO1031 orPRO1122 polypeptide or which enhance or interfere with the function ofthe PRO1031 or PRO1122 polypeptide in vivo. (c.f., Hodgson,Bio/Technology 2: 19-21 (1991)).

In one approach, the three-dimensional structure of the PRO1031 orPRO1122 polypeptide, or of a PRO1031 or PRO1122 polypeptide-inhibitorcomplex, is determined by x-ray cystallography, by computer modeling or,most typically, by a combination of the two approaches. Both the shapeand charges of the PRO1031 or PRO1122 must be ascertained to elucidatethe structure and to determine active sties(s) of the molecule. Lessoften, useful information regarding the structure of the PRO1031 orPRO1122 may be gained by modeling based on the structure of homologousproteins. In both cases, relevant structural information is used todesign analogous PRO1031 or PRO1122 polypeptide-like molecules or toidentity efficient inhibitors. Useful examples of rational drug designmay include molecules which have improved activity or stability as shownby Braxton and Wells, Biochemistry 31: 7796-7801 (1992) or which act asinhibitors, agonists, or antagonists of native peptides as shown byAthauda et al., J. Biochem. 113: 742-746 (1993).

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of anti anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen by used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amounts of the PRO1031 orPRO1122 polypeptide may be made available to perform such analyticalstudies as X-ray crystallography. In addition, knowledge of the PRO1031or PRO1122 polypeptide amino acid sequence provided herein will provideguidance to those employing computer modeling techniques in place or inaddition to x-ray crystallography.

Example 16 Articular cartilage Explant Assay

Introduction:

As mentioned previously, IL-17 is likely to play a role in theinitiation or maintenance of the proinflammatory response. IL-17 is acytokine expressed by CD4⁺ T_(h) cells and induces the secretion ofproinflammatory and hematopoietic cytokines (e.g., IL-1β, TNF-α, IL-6,IL-8, GM-CSF. Aarvak et al., J. Immunol 162: 1246-1251 (1999); Fossiezet al., J. Exp. Med. 183: 2593-2603 (1996); Jovanovic et al, J. Immunol.160: 3513-3521 (1998) in a number of cell types including synoviocytesand macrophages. In the presence of IL-17, fibroblasts sustain sustainthe proliferation of CD34+ hematopoietic progenitors and induce theirpreferential maturation into neutrophils. As a result, Il-17 mayconstitute an early initiator of the T cell-dependent inflammatoryreaction and be part of the cytokine network which bridges the immunesystem to hematopoiesis.

Expression of IL-17 has been found in the synovium of patients withrheumatoid arthritis, psoriatic arthritis, or osteoarthritis, but not innormal joint tissues. IL-17 can synergize with the monocyte-derived,proinflammatory cytokines IL-1β or TNF-α to induce IL-6 and GM-CSF. Byacting directly on synoviocytes, IL-17 could enhance secretion ofproinflammatory cytokines in vivo and thus exacerbate joint inflammationand destruction.

To further understand the possible role of IL-17, Applicants have testedthe effects of IL-17 on cartilage matrix metabolism. In light of theknown catabolic effects of nitric oxide (NO) on cartilage, and theexistence of high levels of NO in arthritic joints, NO production wasalso measured.

Methods:

Articular cartilage explants: The metacarpophalangeal joint of a 4-6month old female pigs was aseptically dissected, and articular cartilageis removed by free-hand slicing in a careful manner so as to avoid theunderlying bone. The cartilage was minced and cultured in bulk for atleast 24 hours in a humidified atmosphere of 95% air 5% CO₂ in serumfree (SF) media (DME/F121:1) with 0.1% BSA and antibiotics. Afterwashing three times, approximately 80 mg of articular cartilage wasaliquoted into micronics tubes and incubated for at least 24 hours inthe above SF media. Test proteins were then added at 1% either alone orin combination with IL-1α (10 ng/ml) (SEQ ID NO:25). Media was harvestedand changed at various timepoints (0, 24, 48, 72 hours) and assayed forproteoglycan content using the 1,9-dimethyl-methylene blue (DMB)colorimetric assay described in Farndale and Buttle, Biochem. Biophys.Acta 883: 173-177 (1985). After labeling (overnight) with ³⁵S-sulfur,the tubes were weighed to determine the amount of tissue. Following anovernight digestion, the amount of proteoglycan remaining in the tissueas well as proteoglycan synthesis (³⁵S-incorporation) is determined.

Measurement of NO production: The assay is based on the principle that2,3-diaminonapthalene (DAN) reacts with nitrite under acidic conditionsto form 1-(H)-naphthotriazole, a flourescent product. As NO is quicklymetabolized into nitrite (NO₂ ⁻¹) and nitrate (NO₃ ⁻¹), detection ofnitrite, is one means of detecting (albeit undercounting) the actual NOproduced. 10 μL of DAN (0.05 mg. mL in 0.62M HCl) is added to 100 μL ofsample (cell culture supernatant), mixed, and incubated at roomtemperature for 10-20 minutes. Reaction is terminated with 5 mL of 2.8NNaOH. Formation of 2,3-diaminonaphthotriazole was measured using aCytoflor flourescent plate reader with excitation at 360 nm and emissionread at 450 nm. For optimal measurement of flourescent intensity, blackplates with clear bottoms were used.

Results and Discussion:

IL-17 (SEQ ID NO:11) was observed to both increase the release of anddecrease the synthesis of proteoglycans (FIG. 13). Moreover, this effectwas additive to the effect observed from IL-1α. (FIG. 13) (SEQ IDNO:25). The effects of IL-17 are not mediated by the production ofnitric oxide, nor does inhibition of nitric oxide release augment matrixbreakdown. UNQ561 (SEQ ID NO:3) increases matrix breakdown and inhibitsmatrix synthesis. Thus, expression of PRO1122 is likely to be associatedwith degenerative cartilagenous disorders. On the other hand, UNQ516(SEQ ID NO:1) increases matrix synthesis and inhibits nitric oxiderelease by articular cartilage explants.

In conclusion, IL-17 likely contributes to loss of articular cartilagein arthritic joints, and thus inhibition of its activity might limitinflammation and cartilage destruction. IL-1 and IL-17 have similar yetdistinct activities due to their use of different receptors andoverlapping downstream signaling mechanisms.

Given the findings of the potent catabolic effects of IL-17 on articularcartilage explants and the homology of UNQ516 and UNQ561 to IL-17,antagonists to any or all of these proteins may be useful for thetreatment of inflammatory conditions and cartilage defects such asarthritis. However, our finding that UNQ516 inhibits NO production andenhances matrix synthesis suggests that this protein and agoniststhereof could have beneficial effects within the joint and may thus, inand of itself, be useful for the treatment of the above mentioneddisorders.

Finally, it is well known that growth factors can have biphasic effectsand that diseased tissue can respond differently than normal tissue to agiven factor in vivo. For these reasons, antagonists or agonists (e.g.the proteins themselves) of UNQ 516, UNQ 561, or IL-17, may be usefulfor the treatment of inflammatory conditions and joint disorders such asarthritis.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va. USA20110-2209 (ATCC): Material ATCC Dep. No. Deposit Date DNA59294-1381209866 14 May 1998 DNA62377-1381-1 203546 22 Dec. 1998

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC §122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1-42. (canceled)
 43. An isolated antibody that binds the polypeptide ofSEQ ID NO:3.
 44. The isolated antibody of claim 43 that binds thepolypeptide of SEQ ID NO:3 lacking its associated signal peptide. 45.The isolated antibody of claim 43 which is a monoclonal antibody. 46.The isolated antibody of claim 43 which is a humanized antibody.
 47. Theisolated antibody of claim 43 which is a human antibody.
 48. Theisolated antibody of claim 43 which is a chimeric antibody.
 49. Theisolated antibody of claim 43 which is a single chain antibody.
 50. Theisolated antibody of claim 43 which is an antibody fragment.
 51. Theisolated antibody of claim 43 which is labeled with a detectable moietycapable of directly or indirectly producing a signal.
 52. Apharmaceutical composition comprising the isolated antibody of claim 43and a pharmaceutically acceptable carrier.
 53. An isolated antibody thatbinds the mature polypeptide of SEQ ID NO:3 consisting of amino acidresidues 19-197 of SEQ ID NO:3.
 54. The isolated antibody of claim 53which is a monoclonal antibody.
 55. The isolated antibody of claim 53which is a humanized antibody.
 56. The isolated antibody of claim 53which is a human antibody.
 57. The isolated antibody of claim 53 whichis a chimeric antibody.
 58. The isolated antibody of claim 53 which is asingle chain antibody.
 59. The isolated antibody of claim 53 which is anantibody fragment.
 60. The isolated antibody of claim 53 which islabeled with a detectable moiety capable of directly or indirectlyproducing a signal.
 61. A pharmaceutical composition comprising theisolated antibody of claim 53 and a pharmaceutically acceptable carrier.62. An isolated antibody that binds the polypeptide encoded by the cDNAclone deposited as ATCC deposit number
 203552. 63. The isolated antibodyof claim 62 which is a monoclonal antibody.
 64. The isolated antibody ofclaim 62 which is a humanized antibody.
 65. The isolated antibody ofclaim 62 which is a human antibody.
 66. The isolated antibody of claim62 which is a chimeric antibody.
 67. The isolated antibody of claim 62which is a single chain antibody.
 68. The isolated antibody of claim 62which is an antibody fragment.
 69. The isolated antibody of claim 62which is labeled with a detectable moiety capable of directly orindirectly producing a signal.
 70. A pharmaceutical compositioncomprising the isolated antibody of claim 62 and a pharmaceuticallyacceptable carrier.